@article{wang_highly_2015, title = {Highly sensitive chemiluminescent detection of lead ion based on its displacement of potassium in G-Quadruplex {DNAzyme}}, volume = {140}, pages = {5742--5747}, number = {16}, journal = {The Analyst}, shortjournal = {Analyst}, author = {Wang, Hong and Wang, Dong Mei and Huang, Cheng Zhi}, } @patent{aptamers_pat1, title = {Aptamers and methods for their in vitro selection and uses thereof}, url = {http://www.google.com/patents/US20050142582}, abstract = {The present method is an improved in vitro selection protocol that relies on magnetic separations for {DNA} aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific {TTF}1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.}, urldate = {2015-09-15}, note = {{US}-Klassifikation 435/6.12, 536/24.3, 435/91.2; Internationale Klassifikation C12N15/115, C12Q, C12Q1/68, C07H21/04, C12P19/34; Unternehmensklassifikation C12N2320/13, C12N2330/30, C12Q1/6811, C12N2310/16, C12N15/115; Europäische Klassifikation C12N15/115} } @patent{aptamers_pat_2, title = {Dna aptamers binding the histidine tag and their application}, url = {http://www.google.com/patents/WO2014185802A1}, abstract = {A {DNA} aptamer was obtained which has an affinity for His-tag, and contains a nucleotide sequence selected from {SEQ} {ID} No. 1 and {SEQ} {ID} No. 2, which has clear applications.}, urldate = {2015-09-15}, note = {Internationale Klassifikation C12N15/115; Unternehmensklassifikation C07K14/001, C07K7/08, C12N15/115, C12N2310/16} } @article{Cottrell2007, abstract = {The hairpin ribozyme is a small catalytic motif found in plant satellite RNAs where it catalyzes a reversible self-cleavage reaction during processing of replication intermediates. Crystallographic studies of hairpin ribozymes have provided high resolution views of the RNA functional groups that comprise the active site and stimulated biochemical studies that probed the contributions of nucleobase functional groups to catalytic chemistry. The dramatic loss of activity that results from perturbation of active site architecture points to the importance of positioning and orientation in catalytic rate acceleration. The current study focuses on the network of noncovalent interactions that align nucleophilic and leaving group oxygens in the orientation required for the S(N)2-type reaction mechanism and orient the active site nucleobases near the reactive phosphate to facilitate catalytic chemistry. Nucleotide modifications that alter or eliminate individual hydrogen bonding partners had different effects on the activation barrier to catalysis, the stability of ribozyme complexes in the ground state, and the internal equilibrium between cleavage and ligation of bound products. Furthermore, substitution of hydrogen bond donors and acceptors with seemingly equivalent pairs sometimes had very different functional consequences. These biochemical analyses augment high resolution structural information to provide insights into the functional significance of active site architecture.}, author = {Cottrell, Joseph W. and Kuzmin, Yaroslav I. and Fedor, Martha J.}, doi = {10.1074/jbc.M700451200}, file = {:root/Downloads/J. Biol. Chem.-2007-Cottrell-13498-507.pdf:pdf}, issn = {00219258}, journal = {Journal of Biological Chemistry}, number = {18}, pages = {13498--13507}, pmid = {17351263}, title = {{Functional analysis of hairpin ribozyme active site architecture}}, volume = {282}, year = {2007} } @article{Diegelman1998, abstract = {A simple new strategy for the in vitro synthesis of circular RNAs and hairpin ribozymes is described. Circular single-strand DNA oligonucleotides 67-79 nt in length are constructed to encode both hairpin ribozyme sequences and ribozyme-cleavable sequences. In vitro transcription of these small circles by Escherichia coli RNA polymerase produces long repeating RNAs by a rolling circle mechanism. These repetitive RNAsundergo self-processing, eventually yielding unit length circular and linear RNAs as the chief products. The transcription is efficient despite the absence of promoter sequences, with RNA being produced in up to 400 times the amount of DNA circle used. It is shown that the linear monomeric hairpin ribozymes are active in cleaving RNA targets in trans , including one from HIV-1. Several new findings are established: (i) that rolling circle transcription can be extended to the synthesis of catalytic RNAs outside the hammerhead ribozyme motif; (ii) that rolling circle transcription is potentially a very simple and useful strategy for the generation of circular RNAs in preparative amounts; and (iii) that self-processed hairpin ribozymes can be catalytically active in trans despite the presence of self-binding domains.}, author = {Diegelman, Amy M. and Kool, Eric T.}, doi = {10.1093/nar/26.13.3235}, file = {:root/Downloads/Nucl. Acids Res.-1998-Diegelman-3235-41.pdf:pdf}, isbn = {0305-1048 (Print) 0305-1048 (Linking)}, issn = {03051048}, journal = {Nucleic Acids Research}, number = {13}, pages = {3235--3241}, pmid = {9628924}, title = {{Generation of circular RNAs and trans-cleaving catalytic RNAs by rolling transcription of circular DNA oligonucleotides encoding hairpin ribozymes}}, volume = {26}, year = {1998} } @article{Drude2011, abstract = {Application of ribozymes for knockdown of RNA targets requires the identification of suitable target sites according to the consensus sequence. For the hairpin ribozyme, this was originally defined as Y⁻² N⁻¹ *G+¹ U+² Y+³ B+⁴, with Y = U or C, and B = U, C or G, and C being the preferred nucleobase at positions -2 and +4. In the context of development of ribozymes for destruction of an oncogenic mRNA, we have designed ribozyme variants that efficiently process RNA substrates at U⁻² G⁻¹ *G+¹ U+² A+³ A+⁴ sites. Substrates with G⁻¹ *G+¹ U+² A+³ sites were previously shown to be processed by the wild-type hairpin ribozyme. However, our study demonstrates that, in the specific sequence context of the substrate studied herein, compensatory base changes in the ribozyme improve activity for cleavage (eight-fold) and ligation (100-fold). In particular, we show that A+³ and A+⁴ are well tolerated if compensatory mutations are made at positions 6 and 7 of the ribozyme strand. Adenine at position +4 is neutralized by G⁶ →U, owing to restoration of a Watson-Crick base pair in helix 1. In this ribozyme-substrate complex, adenine at position +3 is also tolerated, with a slightly decreased cleavage rate. Additional substitution of A⁷ with uracil doubled the cleavage rate and restored ligation, which was lost in variants with A⁷, C⁷ and G⁷. The ability to cleave, in conjunction with the inability to ligate RNA, makes these ribozyme variants particularly suitable candidates for RNA destruction.}, author = {Drude, Irene and Strahl, Anne and Galla, Daniel and M\"{u}ller, Oliver and M\"{u}ller, Sabine}, doi = {10.1111/j.1742-4658.2010.07983.x}, file = {:root/Downloads/544022e90cf21227a11ba527.pdf:pdf}, isbn = {1742-4658 (Electronic)$\backslash$n1742-464X (Linking)}, issn = {1742464X}, journal = {FEBS Journal}, keywords = {RNA,cleavage,hairpin ribozyme,kinetics,ligation}, number = {4}, pages = {622--633}, pmid = {21199369}, title = {{Design of hairpin ribozyme variants with improved activity for poorly processed substrates}}, volume = {278}, year = {2011} } @article{Drude2007, abstract = {Over the past two decades, the structure and mechanism of catalytic RNA have been extensively studied; now ribozymes are understood well enough to turn them into useful tools. After we have demonstrated the twin ribozyme mediated insertion of additional nucleotides into a predefined position of a suitable substrate RNA, we here show that a similar type of twin ribozyme is also capable of mediating the opposite reaction: the site-specific removal of nucleotides. In particular, we have designed a twin ribozyme that supports the deletion of four uridine residues from a given RNA substrate. This reaction is a kind of RNA recombination that in the specific context of gene therapy mimics, at the level of RNA, the correction of insertion mutations. As a result of the twin ribozyme driven reaction, 17\% of substrate are converted into the four nucleotides shorter product RNA. © 2007 Elsevier Inc. All rights reserved.}, author = {Drude, Irene and Vaul\'{e}on, St\'{e}phanie and M\"{u}ller, Sabine}, doi = {10.1016/j.bbrc.2007.08.135}, file = {:root/Downloads/1-s2.0-S0006291X07017780-main.pdf:pdf}, issn = {0006291X}, journal = {Biochemical and Biophysical Research Communications}, keywords = {Catalytic RNA,Gene therapy,Insertion mutation,RNA editing,RNA engineering,RNA repair,Rational design,Ribozyme}, number = {1}, pages = {24--29}, pmid = {17825791}, title = {{Twin ribozyme mediated removal of nucleotides from an internal RNA site}}, volume = {363}, year = {2007} } @article{Fujitani1993, abstract = {The hairpin ribozyme cleaves a phosphodiester bond at the 5' side of a 5'GUC3' sequence of an RNA with high efficiency. An RNA having a 5'GUA3' sequence instead of the GUC sequence is a poor substrate for this ribozyme. Here, we show that this is indeed so in a trans-acting ribozyme system, but in a cis-acting ribozyme system this ribozyme cleaves the 5' side of a GUA sequence as efficiently as the wild-type cleaves the GUC sequence. One base substitution in the ribozyme also affected the target-site specificity in the cis-acting system.}, author = {Fujitani, K. and Sasaki-Tozawa, N. and Kikuchi, Y.}, doi = {10.1016/0014-5793(93)80316-M}, file = {:root/Downloads/1-s2.0-001457939380316M-main.pdf:pdf}, issn = {00145793}, journal = {FEBS Letters}, keywords = {Arabis mosaic virus,catalytic RNA,chicory yellow mottle virus,satellite RNA,tobacco ringspot virus}, number = {1-2}, pages = {155--158}, pmid = {8405396}, title = {{Different target-site specificities of the hairpin ribozyme in cis and trans cleavages}}, volume = {331}, year = {1993} } @article{Hammann2012, abstract = {The hammerhead ribozyme is a small catalytic RNA motif capable of endonucleolytic (self-) cleavage. It is composed of a catalytic core of conserved nucleotides flanked by three helices, two of which form essential tertiary interactions for fast self-scission under physiological conditions. Originally discovered in subviral plant pathogens, its presence in several eukaryotic genomes has been reported since. More recently, this catalytic RNA motif has been shown to reside in a large number of genomes. We review the different approaches in discovering these new hammerhead ribozyme sequences and discuss possible biological functions of the genomic motifs.}, author = {Hammann, C. and Luptak, a. and Perreault, J. and de la Pena, M.}, doi = {10.1261/rna.031401.111}, file = {:root/Downloads/871.pdf:pdf}, isbn = {1469-9001 (Electronic)$\backslash$r1355-8382 (Linking)}, issn = {1355-8382}, journal = {Rna}, keywords = {catalytic rna,database searches,homology,retrotransposons,structure}, number = {5}, pages = {871--885}, pmid = {22454536}, title = {{The ubiquitous hammerhead ribozyme}}, volume = {18}, year = {2012} } @article{Heldenbrand2014, author = {Heldenbrand, Hugh and Janowski, Pawel a and Giambas, George and Giese, Timothy J and Wedekind, Joseph E and York, Darrin M}, file = {:root/Downloads/ja500180q.pdf:pdf}, journal = {Journal of the American Chemical Society}, pages = {8--11}, title = {{from Molecular Simulations along the Reaction Path}}, year = {2014} } @article{Ivanov2005, abstract = {In recent years major progress has been made in elucidating the mechanism and structure of catalytic RNA molecules, and we are now beginning to understand ribozymes well enough to turn them into useful tools. Work in our laboratory has focused on the development of twin ribozymes for site-specific RNA sequence alteration. To this end, we followed a strategy that relies on the combination of two ribozyme units into one molecule (hence dubbed twin ribozyme). Here, we present reverse-joined hairpin ribozymes that are structurally optimized and which, in addition to cleavage, catalyse efficient RNA ligation. The most efficient variant ligated its appropriate RNA substrate with a single turnover rate constant of 1.1 min(-1) and a final yield of 70\%. We combined a reverse-joined hairpin ribozyme with a conventional hairpin ribozyme to create a twin ribozyme that mediates the insertion of four additional nucleotides into a predetermined position of a substrate RNA, and thus mimics, at the RNA level, the repair of a short deletion mutation; 17\% of the initial substrate was converted to the insertion product.}, author = {Ivanov, Sergei a. and Vaul\'{e}on, St́phanie and M\"{u}ller, Sabine}, doi = {10.1111/j.1742-4658.2005.04865.x}, file = {:root/Downloads/j.1742-4658.2005.04865.x(1).pdf:pdf}, issn = {1742464X}, journal = {FEBS Journal}, keywords = {RNA catalysis,RNA ligation,Rational design,Sequence alteration,Twin ribozyme}, number = {17}, pages = {4464--4474}, pmid = {16128815} } @article{Balbo2007, author = {Balbo, P. B. and Bohm, A.}, title = {Mechanism of poly(A) polymerase: structure of the enzyme-MgATP-RNA ternary complex and kinetic analysis}, journal = {Structure}, volume = {15}, number = {9}, pages = {1117-31}, note = {Balbo, Paul B Bohm, Andrew eng GM 065972/GM/NIGMS NIH HHS/ R01 GM065972/GM/NIGMS NIH HHS/ R01 GM065972-05/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural London, England : 1993 2007/09/14 09:00 Structure. 2007 Sep;15(9):1117-31.}, abstract = {We report the 1.8 A structure of yeast poly(A) polymerase (PAP) trapped in complex with ATP and a five residue poly(A) by mutation of the catalytically required aspartic acid 154 to alanine. The enzyme has undergone significant domain movement and reveals a closed conformation with extensive interactions between the substrates and all three polymerase domains. Both substrates and 31 buried water molecules are enclosed within a central cavity that is open at both ends. Four PAP mutants were subjected to detailed kinetic analysis, and studies of the adenylyltransfer (forward), pyrophosphorolysis (reverse), and nucleotidyltransfer reaction utilizing CTP for the mutants are presented. The results support a model in which binding of both poly(A) and the correct nucleotide, MgATP, induces a conformational change, resulting in formation of a stable, closed enzyme state. Thermodynamic considerations of the data are discussed as they pertain to domain closure, substrate specificity, and catalytic strategies utilized by PAP.}, keywords = {Adenosine Triphosphate/*metabolism Catalysis Kinetics Models, Molecular Mutagenesis, Site-Directed Polynucleotide Adenylyltransferase/chemistry/genetics/*metabolism Protein Conformation RNA/chemistry/*metabolism}, ISSN = {0969-2126 (Print) 0969-2126 (Linking)}, DOI = {10.1016/j.str.2007.07.010}, url = {http://www.ncbi.nlm.nih.gov/pubmed/17850751}, year = {2007}, type = {Journal Article} } @article{Baugh2000, author = {Baugh, C. and Grate, D. and Wilson, C.}, title = {2.8 angstrom crystal structure of the malachite green aptamer}, journal = {Journal of Molecular Biology}, volume = {301}, number = {1}, pages = {117-128}, note = {343MC Times Cited:90 Cited References Count:52}, abstract = {Previous in vitro selection experiments identified an RNA aptamer that recognizes the chromophore malachite green (MG) with a high level of affinity, and which undergoes site-specific cleavage following laser irradiation. To understand the mechanism by which this RNA folds to recognize specifically its ligand and the structural basis for chromophore-assisted laser inactivation, we have determined the 2.8 Angstrom crystal structure of the aptamer bound to tetramethylrosamine (TMR), a high-affinity MG analog. The ligand-binding site is defined by an asymmetric internal loop, flanked by a pair of helices. A U-turn and several non-canonical base interactions stabilize the folding of loop nucleotides around the TMR. The aptamer utilizes several tiers of stacked nucleotides arranged in pairs, triples, and a novel base quadruple to effectively encapsulate the ligand. Even in the absence of specific stabilizing hydrogen bonds, discrimination between related fluorophores and chromophores is possible due to tight packing in the RNA binding pocket, which severely limits the size and shape of recognized ligands. The site of laser-induced cleavage lies relatively far from the bound TMR (similar to 15 Angstrom). The unusual backbone conformation of the cleavage site nucleotide and its high level of solvent accessibility may, combine to allow preferential reaction with freely diffusing hydroxyl radicals generated at the bound ligand. Several observations, however, favor alternative mechanisms for cleavage, such as conformational changes in the aptamer or long-range electron transfer between the bound ligand and the cleavage site nucleotide. (C) 2000 Academic Press.}, keywords = {in vitro selection tetramethylrosamine base quadruple chromophore-assisted laser inactivation assisted laser inactivation ribosomal-rna binding rna DNA recognition resolution complex discrimination selection molecules}, ISSN = {0022-2836}, DOI = {10.1006/jmbi.2000.3951}, url = {://WOS:000088705300010}, year = {2000}, type = {Journal Article} } @article{Beckert2011, author = {Beckert, B. and Masquida, B.}, title = {Synthesis of RNA by in vitro transcription}, journal = {Methods Mol Biol}, volume = {703}, pages = {29-41}, note = {Beckert, Bertrand Masquida, Benoit eng Clifton, N.J. 2010/12/03 06:00 Methods Mol Biol. 2011;703:29-41. doi: 10.1007/978-1-59745-248-9_3.}, abstract = {In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases in mug to mg quantities. It is based on the engineering of a template that includes a bacteriophage promoter sequence (e.g. from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase. In vitro transcripts are used in analytical techniques (e.g. hybridization analysis), structural studies (for NMR and X-ray crystallography), in biochemical and genetic studies (e.g. as antisense reagents), and as functional molecules (ribozymes and aptamers).}, keywords = {Bacteriophage T7/genetics Base Sequence DNA-Directed RNA Polymerases/genetics In Vitro Techniques Molecular Biology/*methods Molecular Sequence Data Promoter Regions, Genetic/genetics RNA/*chemical synthesis *Transcription, Genetic}, ISSN = {1940-6029 (Electronic) 1064-3745 (Linking)}, DOI = {10.1007/978-1-59745-248-9_3}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21125481}, year = {2011}, type = {Journal Article} } @article{Bauxbaum2015, author = {Buxbaum, A. R. and Haimovich, G. and Singer, R. H.}, title = {In the right place at the right time: visualizing and understanding mRNA localization}, journal = {Nature Reviews Molecular Cell Biology}, volume = {16}, number = {2}, pages = {95-109}, note = {Ca1ad Times Cited:5 Cited References Count:176}, abstract = {The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization.}, keywords = {yeast saccharomyces-cerevisiae green fluorescent protein in-vitro reconstitution zipcode-binding protein actin gene-expression xenopus-oocytes hippocampal-neurons endoplasmic-reticulum dendritic transport particle tracking}, ISSN = {1471-0072}, DOI = {10.1038/nrm3918}, url = {://WOS:000348643800010}, year = {2015}, type = {Journal Article} } @article{Dean2014, author = {Dean, K. M. and Palmer, A. E.}, title = {Advances in fluorescence labeling strategies for dynamic cellular imaging}, journal = {Nature Chemical Biology}, volume = {10}, number = {7}, pages = {512-523}, note = {Aj7iu Times Cited:22 Cited References Count:96}, abstract = {Synergistic advances in optical physics, probe design, molecular biology, labeling techniques and computational analysis have propelled fluorescence imaging into new realms of spatiotemporal resolution and sensitivity. This review aims to discuss advances in fluorescent probes and live-cell labeling strategies, two areas that remain pivotal for future advances in imaging technology. Fluorescent protein- and bio-orthogonal-based methods for protein and RNA imaging are discussed as well as emerging bioengineering techniques that enable their expression at specific genomic loci (for example, CRISPR and TALENs). Important attributes that contribute to the success of each technique are emphasized, providing a guideline for future advances in dynamic live-cell imaging.}, keywords = {directed tosyl chemistry zinc-finger nucleases living cells in-vivo superresolution microscopy bacterial phytochrome stokes shift live cells protein rna}, ISSN = {1552-4450}, DOI = {10.1038/Nchembio.1556}, url = {://WOS:000337871200008}, year = {2014}, type = {Journal Article} } @article{Fernandey-Suarez2008, author = {Fernandez-Suarez, M. and Ting, A. Y.}, title = {Fluorescent probes for super-resolution imaging in living cells}, journal = {Nat Rev Mol Cell Biol}, volume = {9}, number = {12}, pages = {929-43}, note = {Fernandez-Suarez, Marta Ting, Alice Y eng Review England 2008/11/13 09:00 Nat Rev Mol Cell Biol. 2008 Dec;9(12):929-43. doi: 10.1038/nrm2531. Epub 2008 Nov 12.}, abstract = {In 1873, Ernst Abbe discovered that features closer than approximately 200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1-5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.}, keywords = {Animals Carbocyanines/metabolism Cells/*metabolism Coloring Agents/metabolism Diagnostic Imaging/*methods Fluorescent Dyes/*metabolism Green Fluorescent Proteins/metabolism Luminescent Agents/metabolism Microscopy, Fluorescence/*methods}, ISSN = {1471-0080 (Electronic) 1471-0072 (Linking)}, DOI = {10.1038/nrm2531}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19002208}, year = {2008}, type = {Journal Article} } @book{Hartmann2009, author = {Hartmann, Roland K.}, title = {Handbook of RNA biochemistry}, publisher = {Wiley-VCH}, address = {Weinheim}, edition = {1st student}, note = {2009499142 edited by Roland K. Hartmann ... [et al.]. ill. (some col.) ; 25 cm. Includes bibliographical references and index.}, keywords = {rna.}, pages = {xliii, 931 p.}, ISBN = {9783527325344 (pbk. alk. paper)}, year = {2009}, type = {Book} } @article{Höfer2013, author = {Höfer, K. and Langejürgen, L. V. and Jäschke, A.}, title = {Universal aptamer-based real-time monitoring of enzymatic RNA synthesis}, journal = {J Am Chem Soc}, volume = {135}, number = {37}, pages = {13692-4}, note = {Hofer, Katharina Langejurgen, Lisa V Jaschke, Andres eng 2013/09/03 06:00 J Am Chem Soc. 2013 Sep 18;135(37):13692-4. doi: 10.1021/ja407142f. Epub 2013 Sep 4.}, abstract = {In vitro transcription is an essential laboratory technique for enzymatic RNA synthesis. Unfortunately, no methods exist for analyzing quality and quantity of the synthesized RNA while the transcription proceeds. Here we describe a simple, robust, and universal system for monitoring and quantifying the synthesis of any RNA in real time without interference from abortive transcription byproducts. The distinguishing feature is a universal fluorescence module (UFM), consisting of the eGFP-like Spinach aptamer and a highly active hammerhead ribozyme, which is appended to the RNA of interest (ROI). In the transcription mixture, the primary transcript is cleaved rapidly behind the ROI, thereby releasing always the same UFM, independent of the ROI sequence, polymerase, or promoter used. The UFM binds to the target of the Spinach aptamer, the fluorogenic dye DFHBI, and thereby induces a strong fluorescence signal. This design allows real-time quantification, standardization, parallelization, and high-throughput screening.}, keywords = {Aptamers, Nucleotide/*chemical synthesis/chemistry *Biological Assay Exodeoxyribonucleases/*metabolism Fluorescence}, ISSN = {1520-5126 (Electronic) 0002-7863 (Linking)}, DOI = {10.1021/ja407142f}, url = {http://www.ncbi.nlm.nih.gov/pubmed/23991672}, year = {2013}, type = {Journal Article} } @article{Kellenberger2015, author = {Kellenberger, C. A. and Chen, C. and Whiteley, A. T. and Portnoy, D. A. and Hammond, M. C.}, title = {RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP}, journal = {Journal of the American Chemical Society}, volume = {137}, number = {20}, pages = {6432-6435}, note = {Cj3kv Times Cited:0 Cited References Count:24}, abstract = {Cyclic di-AMP (cdiA) is a second messenger predicted, to be widespread in Gram-positive bacteria, some Gram-negative bacteria, and Archaea. In the human pathogen Listeria monocytogenes, cdiA is an essential molecule that regulates metabolic function and cell wall homeostasis, and decreased levels of cdiA result in increased antibiotic susceptibility. We have generated fluorescent biosensors for cdiA through fusion of the Spinach2 aptamer to ligand-binding domains of cdiA riboswitches. The biosensor was used to visualize intra-cellular cdiA levels in live L. monocytogenes strains and to determine the catalytic domain of the phosphodiesterase PdeA. Furthermore; a flow cytometry assay based On this biosensor was used to screen for diadenylate cyclase activity and confirmed the enzymatic activity of DisA-like proteins from Clostridium difficile and Methanocaldococcus jannaschii. Thus, We have expanded the development of RNA-based biosensors for in vivo metabolite imaging in Gram-positive bacteria and have validated the first dinucleotide cyclase from Archaea.}, keywords = {bacterial 2nd-messenger ydao riboswitch small molecules nucleotides virulence reveals enters sense gmp}, ISSN = {0002-7863}, DOI = {10.1021/jacs.5b00275}, url = {://WOS:000355383500003}, year = {2015}, type = {Journal Article} } @article{Kellenberber2013, author = {Kellenberger, C. A. and Wilson, S. C. and Sales-Lee, J. and Hammond, M. C.}, title = {RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messengers Cyclic di-GMP and Cyclic AMP-GMP}, journal = {Journal of the American Chemical Society}, volume = {135}, number = {13}, pages = {4906-4909}, note = {121QM Times Cited:32 Cited References Count:24}, abstract = {Cyclic dinucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed RNA-based fluorescent biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-1 riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic dinucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.}, keywords = {escherichia-coli ligand-binding riboswitch diguanylate magnesium bacteria cgmp DNA}, ISSN = {0002-7863}, DOI = {10.1021/ja311960g}, url = {://WOS:000317259300002}, year = {2013}, type = {Journal Article} } @article{Paige2011, author = {Paige, J. S. and Wu, K. Y. and Jaffrey, S. R.}, title = {RNA mimics of green fluorescent protein}, journal = {Science}, volume = {333}, number = {6042}, pages = {642-6}, note = {Paige, Jeremy S Wu, Karen Y Jaffrey, Samie R eng NS064516/NS/NINDS NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ R01 NS064516-03/NS/NINDS NIH HHS/ T32 CA062948/CA/NCI NIH HHS/ T32 CA062948-14/CA/NCI NIH HHS/ T32CA062948/CA/NCI NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't New York, N.Y. 2011/07/30 06:00 Science. 2011 Jul 29;333(6042):642-6. doi: 10.1126/science.1207339.}, abstract = {Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here, we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex, termed Spinach, resembles enhanced GFP and emits a green fluorescence comparable in brightness with fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.}, keywords = {Aptamers, Nucleotide/*chemistry/*metabolism Benzyl Compounds/*chemistry/*metabolism Biomimetics Cell Nucleus/metabolism Cytoplasmic Granules/metabolism Cytosol/metabolism *Fluorescence Green Fluorescent Proteins/*chemistry HEK293 Cells Humans Imidazolines/*chemistry/*metabolism Molecular Mimicry Nucleic Acid Conformation Photobleaching Protein Binding RNA, Untranslated/metabolism SELEX Aptamer Technique Spectrometry, Fluorescence Sucrose/pharmacology}, ISSN = {1095-9203 (Electronic) 0036-8075 (Linking)}, DOI = {10.1126/science.1207339}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21798953}, year = {2011}, type = {Journal Article} } @article{Pothoulakis2014, author = {Pothoulakis, G. and Ceroni, F. and Reeve, B. and Ellis, T.}, title = {The spinach RNA aptamer as a characterization tool for synthetic biology}, journal = {ACS Synth Biol}, volume = {3}, number = {3}, pages = {182-7}, note = {Pothoulakis, Georgios Ceroni, Francesca Reeve, Benjamin Ellis, Tom eng Research Support, Non-U.S. Gov't 2013/09/03 06:00 ACS Synth Biol. 2014 Mar 21;3(3):182-7. doi: 10.1021/sb400089c. Epub 2013 Sep 13.}, abstract = {Characterization of genetic control elements is essential for the predictable engineering of synthetic biology systems. The current standard for in vivo characterization of control elements is through the use of fluorescent reporter proteins such as green fluorescent protein (GFP). Gene expression, however, involves not only protein production but also the production of mRNA. Here, we present the use of the Spinach aptamer sequence, an RNA mimic of GFP, as a tool to characterize mRNA expression in Escherichia coli. We show how the aptamer can be incorporated into gene expression cassettes and how co-expressing it with a red fluorescent protein (mRFP1) allows, for the first time, simultaneous measurement of mRNA and protein levels from engineered constructs. Using flow cytometry, we apply this tool here to evaluate ribosome binding site sequences and promoters and use it to highlight the differences in the temporal behavior of transcription and translation.}, keywords = {Aptamers, Nucleotide/*genetics/metabolism Binding Sites Escherichia coli/genetics/metabolism Genetic Engineering/*methods Luminescent Proteins/genetics/metabolism RNA/*genetics/metabolism RNA, Bacterial/genetics/metabolism Spinacia oleracea/*genetics Synthetic Biology/*methods}, ISSN = {2161-5063 (Electronic) 2161-5063 (Linking)}, DOI = {10.1021/sb400089c}, url = {http://www.ncbi.nlm.nih.gov/pubmed/23991760}, year = {2014}, type = {Journal Article} } @article{Sassanfar1993, author = {Sassanfar, M. and Szostak, J. W.}, title = {An Rna Motif That Binds Atp}, journal = {Nature}, volume = {364}, number = {6437}, pages = {550-553}, note = {Lq667 Times Cited:389 Cited References Count:17}, abstract = {RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules1,2. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.}, keywords = {molecular recognition receptor}, ISSN = {0028-0836}, DOI = {Doi 10.1038/364550a0}, url = {://WOS:A1993LQ66700062}, year = {1993}, type = {Journal Article} } @article{Sastry1997, author = {Sastry, S. S. and Ross, B. M.}, title = {Nuclease activity of T7 RNA polymerase and the heterogeneity of transcription elongation complexes}, journal = {J Biol Chem}, volume = {272}, number = {13}, pages = {8644-52}, note = {Sastry, S S Ross, B M eng Research Support, Non-U.S. Gov't 1997/03/28 J Biol Chem. 1997 Mar 28;272(13):8644-52.}, abstract = {We have discovered that T7 RNA polymerase, purified to apparent homogeneity from overexpressing Escherichia coli cells, possesses a DNase and an RNase activity. Mutations in the active center of T7 RNA polymerase abolished or greatly decreased the nuclease activity. This nuclease activity is specific for single-stranded DNA and RNA oligonucleotides and does not manifest on double-stranded DNAs. Under the conditions of promoter-driven transcription on double-stranded DNA, no nuclease activity was observed. The nuclease attacks DNA oligonucleotides in mono- or dinucleotide steps. The nuclease is a 3' to 5' exonuclease leaving a 3'-OH end, and it degrades DNA oligonucleotides to a minimum size of 3 to 5 nucleotides. It is completely dependent on Mg2+. The T7 RNA polymerase-nuclease is inhibited by T7 lysozyme and heparin, although not completely. In the presence of rNTPs, the nuclease activity is suppressed but an unusual 3'-end-initiated polymerase activity is unmasked. RNA from isolated pre-elongation and elongation complexes arrested by a psoralen roadblock or naturally paused at the 3'-end of an oligonucleotide template exhibited evidence of nuclease activity. The nuclease activity of T7 RNA polymerase is unrelated to pyrophosphorolysis. We propose that the nuclease of T7 RNA polymerase acts only in arrested or paused elongation complexes, and that in combination with the unusual 3'-end polymerizing activity, causes heterogeneity in elongation complexes. Additionally, during normal transcription elongation, the kinetic balance between nuclease and polymerase is shifted in favor of polymerase.}, keywords = {*Bacteriophage T7 Base Sequence DNA, Single-Stranded/metabolism DNA-Directed RNA Polymerases/*metabolism Deoxyribonucleases/metabolism Electrophoresis, Polyacrylamide Gel Enzyme Activation Furocoumarins/metabolism Heparin/pharmacology Magnesium/metabolism Molecular Sequence Data N-Acetylmuramoyl-L-alanine Amidase/metabolism *Peptide Elongation Factors RNA/metabolism Ribonucleases/metabolism Transcription, Genetic Viral Proteins}, ISSN = {0021-9258 (Print) 0021-9258 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/9079696}, year = {1997}, type = {Journal Article} } @article{Strack2013, author = {Strack, R. L. and Disney, M. D. and Jaffrey, S. R.}, title = {A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA}, journal = {Nat Methods}, volume = {10}, number = {12}, pages = {1219-24}, note = {Strack, Rita L Disney, Matthew D Jaffrey, Samie R eng F32 GM106683/GM/NIGMS NIH HHS/ GM079235/GM/NIGMS NIH HHS/ NS010249/NS/NINDS NIH HHS/ R01 EB010249/EB/NIBIB NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ Research Support, N.I.H., Extramural 2013/10/29 06:00 Nat Methods. 2013 Dec;10(12):1219-24. doi: 10.1038/nmeth.2701. Epub 2013 Oct 27.}, abstract = {Imaging RNA in living cells is a challenging problem in cell biology. One strategy for genetically encoding fluorescent RNAs is to express them as fusions with Spinach, an 'RNA mimic of GFP'. We found that Spinach was dimmer than expected when used to tag constructs in living cells owing to a combination of thermal instability and a propensity for misfolding. Using systematic mutagenesis, we generated Spinach2 that overcomes these issues and can be used to image diverse RNAs. Using Spinach2, we detailed the dynamics of the CGG trinucleotide repeat-containing 'toxic RNA' associated with Fragile X-associated tremor/ataxia syndrome, and show that these RNAs form nuclear foci with unexpected morphological plasticity that is regulated by the cell cycle and by small molecules. Together, these data demonstrate that Spinach2 exhibits improved versatility for fluorescently labeling RNAs in living cells.}, keywords = {Animals Base Sequence COS Cells Cercopithecus aethiops DNA Mutational Analysis Escherichia coli/metabolism Fluorescent Dyes/chemistry Green Fluorescent Proteins/metabolism HEK293 Cells Humans Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Protein Denaturation Protein Folding RNA/*chemistry RNA, Untranslated/*chemistry Temperature *Trinucleotide Repeats}, ISSN = {1548-7105 (Electronic) 1548-7091 (Linking)}, DOI = {10.1038/nmeth.2701}, url = {http://www.ncbi.nlm.nih.gov/pubmed/24162923}, year = {2013}, type = {Journal Article} } @article{Strack2015, author = {Strack, R. L. and Jaffrey, S. R.}, title = {Live-cell imaging of mammalian RNAs with Spinach2}, journal = {Methods Enzymol}, volume = {550}, pages = {129-46}, note = {Strack, Rita L Jaffrey, Samie R eng F32 GM106683/GM/NIGMS NIH HHS/ NS010249/NS/NINDS NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ Research Support, N.I.H., Extramural 2015/01/22 06:00 Methods Enzymol. 2015;550:129-46. doi: 10.1016/bs.mie.2014.10.044. Epub 2015 Jan 6.}, abstract = {The ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of imaging individual RNAs of interest in living cells, each with their own advantages and limitations. This chapter provides an overview of current methods of live-cell RNA imaging, including a detailed discussion of genetically encoded strategies for labeling RNAs in mammalian cells. This chapter then focuses on the development and use of "RNA mimics of GFP" or Spinach technology for tagging mammalian RNAs and includes a detailed protocol for imaging 5S and CGG60 RNA with the recently described Spinach2 tag.}, ISSN = {1557-7988 (Electronic) 0076-6879 (Linking)}, DOI = {10.1016/bs.mie.2014.10.044}, url = {http://www.ncbi.nlm.nih.gov/pubmed/25605384}, year = {2015}, type = {Journal Article} } @article{Tyagi2009, author = {Tyagi, S.}, title = {Imaging intracellular RNA distribution and dynamics in living cells}, journal = {Nat Methods}, volume = {6}, number = {5}, pages = {331-8}, note = {Tyagi, Sanjay eng MH079197/MH/NIMH NIH HHS/ Research Support, N.I.H., Extramural Review 2009/05/01 09:00 Nat Methods. 2009 May;6(5):331-8. doi: 10.1038/nmeth.1321.}, abstract = {Powerful methods now allow the imaging of specific mRNAs in living cells. These methods enlist fluorescent proteins to illuminate mRNAs, use labeled oligonucleotide probes and exploit aptamers that render organic dyes fluorescent. The intracellular dynamics of mRNA synthesis, transport and localization can be analyzed at higher temporal resolution with these methods than has been possible with traditional fixed-cell or biochemical approaches. These methods have also been adopted to visualize and track single mRNA molecules in real time. This review explores the promises and limitations of these methods.}, keywords = {Aptamers, Nucleotide/chemistry Cell Survival Cells/*cytology/*metabolism Cytophotometry/*methods Fluorescent Dyes/chemistry Green Fluorescent Proteins/chemistry/genetics Microscopy, Fluorescence/methods Oligonucleotide Probes/chemistry RNA, Messenger/*analysis/chemistry/*metabolism RNA-Binding Proteins/chemistry/genetics Recombinant Fusion Proteins/chemistry/genetics}, ISSN = {1548-7105 (Electronic) 1548-7091 (Linking)}, DOI = {10.1038/nmeth.1321}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19404252}, year = {2009}, type = {Journal Article} } title = {{Efficient RNA ligation by reverse-joined hairpin ribozymes and engineering of twin ribozymes consisting of conventional and reverse-joined hairpin ribozyme units}}, volume = {272}, year = {2005} } @article{Kath-Schorr2012, abstract = {The catalytic mechanism by which the hairpin ribozyme accelerates cleavage or ligation of the phosphodiester backbone of RNA has been incompletely understood. There is experimental evidence for an important role for an adenine (A38) and a guanine (G8), and it has been proposed that these act in general acid-base catalysis. In this work we show that a large reduction in cleavage rate on substitution of A38 by purine (A38P) can be reversed by replacement of the 5'-oxygen atom at the scissile phosphate by sulfur (5'-PS), which is a much better leaving group. This is consistent with A38 acting as the general acid in the unmodified ribozyme. The rate of cleavage of the 5'-PS substrate by the A38P ribozyme increases with pH log-linearly, indicative of a requirement for a deprotonated base with a relatively high pK(a). On substitution of G8 by diaminopurine, the 5'-PS substrate cleavage rate at first increases with pH and then remains at a plateau, exhibiting an apparent pK(a) consistent with this nucleotide acting in general base catalysis. Alternative explanations for the pH dependence of hairpin ribozyme reactivity are discussed, from which we conclude that general acid-base catalysis by A38 and G8 is the simplest and most probable explanation consistent with all the experimental data.}, author = {Kath-Schorr, Stephanie and Wilson, Timothy J. and Li, Nan Sheng and Lu, Jun and Piccirilli, Joseph a. and Lilley, David M J}, doi = {10.1021/ja3067429}, file = {:root/Downloads/ja3067429.pdf:pdf}, isbn = {1520-5126 (Electronic) 0002-7863 (Linking)}, issn = {00027863}, journal = {Journal of the American Chemical Society}, number = {40}, pages = {16717--16724}, pmid = {22958171}, title = {{General acid-base catalysis mediated by nucleobases in the hairpin ribozyme}}, volume = {134}, year = {2012} } @article{Kazakov2006, abstract = {Although reducing the temperature slows most chemical reactions, freezing can stimulate some reactions by mechanisms that are only partially understood. Here we show that freezing stimulates the self-ligation (circularization) of linear forms of the hairpin ribozyme (HPR) containing 2',3'-cyclic phosphate and 5'-OH termini. Divalent metal ions (M2+) are not required, but monovalent cations and anions at millimolar concentrations can have various effects on this reaction depending on the specific ion. Under optimal conditions, the observed rate of M2+-independent self-ligation reaches a peak (0.04 min(-1)) at -10 degrees C with a yield of -60\% after 1 h. In contrast, no ligation occurs either at above 0 degrees C or in solutions that remain unfrozen when supercooled to subzero temperatures. Under freezing conditions, the cleavage-ligation equilibrium strongly favors ligation. Besides freezing, evaporation of the aqueous solvent as well as the presence of ethanol at levels of 40\% or above can also induce M2+-independent HPR ligation at 25 degrees C. We argue that partial RNA dehydration, which is a common feature of freezing, evaporation, and the presence of ethanol, is a key factor supporting HPR ligation activity at both above- and below-freezing temperatures. In the context of the RNA world hypothesis, freezing-induced ligation is an attractive mechanism by which complex RNAs could have evolved under conditions in which RNA was relatively protected against degradation.}, author = {Kazakov, Sergei a and Balatskaya, Svetlana V and Johnston, Brian H}, doi = {10.1261/rna.2123506}, file = {:root/Downloads/0120446.pdf:pdf}, isbn = {1355-8382 (Print)$\backslash$r1355-8382 (Linking)}, issn = {1355-8382}, journal = {RNA (New York, N.Y.)}, keywords = {dehydration,ethanol,freezing,hairpin ribozyme,ligation,rna world}, number = {3}, pages = {446--456}, pmid = {16495237}, title = {{Ligation of the hairpin ribozyme in cis induced by freezing and dehydration.}}, volume = {12}, year = {2006} } @article{Meli2003, abstract = {Adenine-dependent hairpin ribozymes were isolated by in vitro selection from a degenerated hairpin ribozyme population. Two new adenine-dependent ribozymes catalyze their own reversible cleavage in the presence of free adenine. Both aptamers have Mg(2+) requirements for adenine-assisted cleavage similar to the wild-type hairpin ribozyme. Cleavage kinetics studies in the presence of various other small molecules were compared. The data suggest that adenine does not induce RNA self-cleavage in the same manner for both aptamers. In addition, investigations of pH effects on catalytic rates show that both adenine-dependent aptamers are more active in basic conditions, suggesting that they use new acid/base catalytic strategies in which adenine could be involved directly. The discovery of hairpin ribozymes dependent on adenine for their reversible self-cleavage presents considerable biochemical and evolutionary interests because we show that RNA is able to use exogenous reactive molecules to enhance its own catalytic activity. Such a mechanism may have been a means by which the ribozymes of the RNA world enlarged their chemical repertoire.}, author = {Meli, Marc and Vergne, Jacques and Maurel, Marie Christine}, doi = {10.1074/jbc.M213058200}, file = {:root/Downloads/jbc2003\_usl.pdf:pdf}, issn = {00219258}, journal = {Journal of Biological Chemistry}, number = {11}, pages = {9835--9842}, pmid = {12519767}, title = {{In vitro selection of adenine-dependent hairpin ribozymes}}, volume = {278}, year = {2003} } @article{Paul2002, abstract = {A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent. When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic. A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of itself through an RNA-catalyzed RNA ligation reaction. The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner. This self-replicating system depends on the catalytic nature of the RNA for the generation of copies. A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth. The autocatalytic rate constant was 0.011 min(-1), whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 x 10(-11) M.min(-1). Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates. Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication.}, author = {Paul, Natasha and Joyce, Gerald F}, doi = {10.1073/pnas.202471099}, file = {:root/Downloads/PNAS-2002-Paul-12733-40.pdf:pdf}, isbn = {0027-8424}, issn = {00278424}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, number = {20}, pages = {12733--12740}, pmid = {12239349}, title = {{A self-replicating ligase ribozyme.}}, volume = {99}, year = {2002} } @article{Petkovic2015, author = {Petkovic, Sonja and Badelt, Stefan and Block, Stephan and Flamm, Christoph and Delcea, Mihaela and Hofacker, I V O and M\"{u}ller, Sabine}, doi = {10.1261/rna.047670.114.}, file = {:root/Downloads/RNA-2015-Petkovic-rna.047670.114.pdf:pdf}, keywords = {afm,circularization,computational design,hairpin ribozyme,rna,self-processing}, pages = {1--12}, title = {{Sequence-controlled RNA self-processing : computational design , biochemical analysis , and visualization by AFM}}, year = {2015} } @article{Pinard1999, abstract = {To form a catalytically active complex, the essential nucleotides of the hairpin ribozyme, embedded within the internal loops of the two domains, must interact with one another. Little is known about the nature of these essential interdomain interactions. In the work presented here, we have used recent topographical constraints and other biochemical data in conjunction with molecular modeling (constraint-satisfaction program MC-SYM) to generate testable models of interdomain interactions. Visual analysis of the generated models has revealed a potential interdomain base pair between the conserved guanosine immediately downstream of the reactive phosphodiester (G(+1)) and C(25) within the large domain. We have tested this former model through activity assays, using all 16 combinations of bases at positions +1 and 25. When the standard ribozyme was used, catalytic activity was severely suppressed with substrates containing U(+1), C(+1), or A(+1). Similarly, mutations of the putative pairing partner (C(25) to A(25) or G(25)) reduce activity by several orders of magnitude. The U(25) substitution retains a significant level of activity, consistent with the possible formation of a G.U wobble pair. Strikingly, when combinations of Watson-Crick (or wobble) base pairs were introduced in these positions, catalytic activity was restored, strongly suggesting the existence of the proposed interaction. These results provide a structural basis for the guanosine requirement of this ribozyme and indicate that the hairpin ribozyme can now be engineered to cleave a wider range of RNA sequences.}, author = {Pinard, Robert and Lambert, Dominic and Walter, Nils G. and Heckman, Joyce E. and Major, Fran\c{c}ois and Burke, John M.}, doi = {10.1021/bi992024s}, file = {:root/Downloads/bi992024s.pdf:pdf}, isbn = {0006-2960 (Print) 0006-2960 (Linking)}, issn = {00062960}, journal = {Biochemistry}, number = {49}, pages = {16035--16039}, pmid = {10587425}, title = {{Structural basis for the guanosine requirement of the hairpin ribozyme}}, volume = {38}, year = {1999} } @article{Ruff2012, author = {Ruff, Patrick and Pai, Rekha B. and Storici, Francesca}, doi = {10.5402/2012/939083}, file = {:root/Downloads/939083.pdf:pdf}, issn = {2090-7907}, journal = {ISRN Molecular Biology}, pages = {1--9}, title = {{Real-Time PCR-Coupled CE-SELEX for DNA Aptamer Selection}}, volume = {2012}, year = {2012} } @article{Rupert2001, abstract = {The hairpin ribozyme catalyses sequence-specific cleavage of RNA. The active site of this natural RNA results from the docking of two irregular helices: stems A and B. One strand of stem A harbours the scissile bond. The 2.4 A resolution structure of a hairpin ribozyme-inhibitor complex reveals that the ribozyme aligns the 2'-OH nucleophile and the 5'-oxo leaving group by twisting apart the nucleotides that flank the scissile phosphate. The base of the nucleotide preceding the cleavage site is stacked within stem A; the next nucleotide, a conserved guanine, is extruded from stem A and accommodated by a highly complementary pocket in the minor groove of stem B. Metal ions are absent from the active site. The bases of four conserved purines are positioned potentially to serve as acid-base catalysts. This is the first structure determination of a fully assembled ribozyme active site that catalyses a phosphodiester cleavage without recourse to metal ions.}, author = {Rupert, P B and Ferr\'{e}-D'Amar\'{e}, a R}, doi = {10.1038/35071009}, file = {:root/Downloads/410780a0.pdf:pdf}, isbn = {0028-0836}, issn = {0028-0836}, journal = {Nature}, number = {6830}, pages = {780--786}, pmid = {11298439}, title = {{Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis.}}, volume = {410}, year = {2001} } @article{Song2014, author = {Song, Seongeun and Cho, Yea Seul and Lee, Sung-jae and Hah, Sang Soo}, file = {:root/Downloads/B140912\_2665.pdf:pdf}, keywords = {affinity precipitation,aptamer,his-tagged,immunoprecipitation}, number = {9}, pages = {2665--2668}, title = {{Aptamer-Based Precipitation as an Alternative to the Conventional Immunoprecipitation for Purification of Target Proteins}}, volume = {35}, year = {2014} } @article{Tan2003, abstract = {The natural form of the hairpin ribozyme comprises two major structural elements: a four-way RNA junction and two internal loops carried by adjacent arms of the junction. The ribozyme folds into its active conformation by an intimate association between the loops, and the efficiency of this process is greatly enhanced by the presence of the junction. We have used single-molecule spectroscopy to show that the natural form fluctuates among three distinct states: the folded state and two additional, rapidly interconverting states (proximal and distal) that are inherited from the junction. The proximal state juxtaposes the two loop elements, thereby increasing the probability of their interaction and thus accelerating folding by nearly three orders of magnitude and allowing the ribozyme to fold rapidly in physiological conditions. Therefore, the hairpin ribozyme exploits the dynamics of the junction to facilitate the formation of the active site from its other elements. Dynamic interplay between structural elements, as we demonstrate for the hairpin ribozyme, may be a general theme for other functional RNA molecules.}, author = {Tan, Elliot and Wilson, Timothy J and Nahas, Michelle K and Clegg, Robert M and Lilley, David M J and Ha, Taekjip}, doi = {10.1073/pnas.1233536100}, file = {:root/Downloads/PNAS-2003-Tan-9308-13.pdf:pdf}, isbn = {0027-8424 (Print)$\backslash$r0027-8424 (Linking)}, issn = {0027-8424}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, number = {16}, pages = {9308--9313}, pmid = {12883002}, title = {{A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate.}}, volume = {100}, year = {2003} } @article{Teller2009, abstract = {Engineered nucleic acid hairpin structures are used for the amplified analysis of low-molecular-weight substrates (adenosine monophosphate, AMP) or proteins (lysozyme). The hairpin structures consist of the anti-AMP or antilysozyme aptamer units linked to the horseradish peroxidase (HRP)-mimicking DNAzyme sequence. The HRP-mimicking DNAzyme sequence is protected in a "caged", inactive structure in the stem regions of the respective hairpins, whereas the loop regions include a part of the respective aptamer sequence. The opening of the hairpins by the analytes, AMP or lysozyme, through the formation of the respective analyte-aptamer complexes, results in the self-assembly of the active HRP-mimicking DNAzyme. The DNAzyme catalyzes the H(2)O(2)-mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(2-)) to the colored ABTS(*-), thus providing the amplified optical detection of the respective analytes. The engineered aptamer-DNAzyme hairpin structures reveal significantly improved analytical performance, as compared to analogous fluorophore-quencher-labeled hairpins.}, author = {Teller, Carsten and Shimron, Simcha and Willner, Itamar}, doi = {10.1021/ac901773b}, file = {:root/Downloads/ac901773b.pdf:pdf}, isbn = {1520-6882 (Electronic)$\backslash$n0003-2700 (Linking)}, issn = {00032700}, journal = {Analytical Chemistry}, number = {21}, pages = {9114--9119}, pmid = {19780593}, title = {{Aptamer-DNAzyme hairpins for amplified biosensing}}, volume = {81}, year = {2009} } @article{Wang2013, abstract = {SERS labels are a new class of nanotags for optical detection based on Raman scattering. Central advantages include their spectral multiplexing capacity due to the small line width of vibrational Raman bands, quantification based on spectral intensities, high photostability, minimization of autofluorescence from biological specimens via red to near-infrared (NIR) excitation, and the need for only a single laser excitation line. Current concepts for the rational design and synthesis of SERS labels are summarized in this review. Chemical constituents of SERS labels are the plasmonically active metal colloids for signal enhancement upon resonant laser excitation, organic Raman reporter molecules for adsorption onto the metal surface for identification, and an optional protective shell. Different chemical approaches towards the synthesis of rationally designed SERS labels are highlighted, including also their subsequent bioconjugation.}, author = {Wang, Yuling and Schl\"{u}cker, Sebastian}, doi = {10.1039/c3an36866a}, file = {:root/Downloads/art\%3A10.1023\%2FA\%3A1026644313406.pdf:pdf}, issn = {1364-5528}, journal = {The Analyst}, keywords = {catalytic rna,fluorescent oligonucleotides,reaction kinetics,ribozyme design,rna synthesis}, number = {8}, pages = {2224--2238}, pmid = {23420174}, title = {{Rational design and synthesis of SERS labels.}}, url = {http://www.ncbi.nlm.nih.gov/pubmed/23420174}, volume = {138}, year = {2013} } @article{Zhuang2002, abstract = {We have studied the correlation between structural dynamics and function of the hairpin ribozyme. The enzyme-substrate complex exists in either docked (active) or undocked (inactive) conformations. Using single-molecule fluorescence methods, we found complex structural dynamics with four docked states of distinct stabilities and a strong memory effect where each molecule rarely switches between different docked states. We also found substrate cleavage to be rate-limited by a combination of conformational transitions and reversible chemistry equilibrium. The complex structural dynamics quantitatively explain the heterogeneous cleavage kinetics common to many catalytic RNAs. The intimate coupling of structural dynamics and function is likely a general phenomenon for RNA.}, author = {Zhuang, Xiaowei and Kim, Harold and Pereira, Miguel J B and Babcock, Hazen P and Walter, Nils G and Chu, Steven}, doi = {10.1126/science.1069013}, file = {:root/Downloads/Science1473.pdf:pdf}, isbn = {0036-8075}, issn = {00368075}, journal = {Science (New York, N.Y.)}, number = {5572}, pages = {1473--1476}, pmid = {12029135}, title = {{Correlating structural dynamics and function in single ribozyme molecules.}}, volume = {296}, year = {2002} } @article{Tseng2011, abstract = {Aptamers are short RNA/DNA sequences that are identified through the process of systematic evolution of ligands by exponential enrichment and that bind to diverse biomolecular targets. Aptamers have strong and specific binding through molecular recognition and are promising tools in studying molecular biology. They are recognized as having potential therapeutic and diagnostic clinical applications. The success of the systematic evolution of ligands by exponential enrichment process requires that the RNA/DNA pools used in the process have a sufficient level of sequence diversity and structural complexity. While the systematic evolution of ligands by exponential enrichment technology is well developed, it remains a challenge in the efficient identification of correct aptamers. In this article, we propose a novel information-driven approach to a theoretical design of aptamer templates based solely on the knowledge regarding the biomolecular target structures. We have investigated both theoretically and experimentally the applicability of the proposed approach by considering two specific targets: the serum protein thrombin and the cell membrane phospholipid phosphatidylserine. Both of these case studies support our method and indicate a promising advancement in theoretical aptamer design. In unfavorable cases where the designed sequences show weak binding affinity, these template sequences can be still modified to enhance their affinities without going through the systematic evolution of ligands by exponential enrichment process.}, author = {Tseng, Chih Yuan and Ashrafuzzaman, Md and Mane, Jonathan Y. and Kapty, Janice and Mercer, John R. and Tuszynski, Jack a.}, doi = {10.1111/j.1747-0285.2011.01125.x}, file = {:root/Downloads/j.1747-0285.2011.01125.x.pdf:pdf}, isbn = {1747-0277}, issn = {17470277}, journal = {Chemical Biology and Drug Design}, keywords = {Bioinformatics,Mechanism-based drug design,Molecular recognition,Structure-based drug design}, number = {1}, pages = {1--13}, pmid = {21496214}, title = {{Entropic fragment-based approach to Aptamer design}}, volume = {78}, year = {2011} } @article{Chushak2009, abstract = {In vitro selection of RNA aptamers that bind to a specific ligand usually begins with a random pool of RNA sequences. We propose a computational approach for designing a starting pool of RNA sequences for the selection of RNA aptamers for specific analyte binding. Our approach consists of three steps: (i) selection of RNA sequences based on their secondary structure, (ii) generating a library of three-dimensional (3D) structures of RNA molecules and (iii) high-throughput virtual screening of this library to select aptamers with binding affinity to a desired small molecule. We developed a set of criteria that allows one to select a sequence with potential binding affinity from a pool of random sequences and developed a protocol for RNA 3D structure prediction. As verification, we tested the performance of in silico selection on a set of six known aptamer-ligand complexes. The structures of the native sequences for the ligands in the testing set were among the top 5\% of the selected structures. The proposed approach reduces the RNA sequences search space by four to five orders of magnitude--significantly accelerating the experimental screening and selection of high-affinity aptamers.}, author = {Chushak, Yaroslav and Stone, Morley O.}, doi = {10.1093/nar/gkp408}, file = {:root/Downloads/gkp408.pdf:pdf}, isbn = {1362-4962 (Electronic)$\backslash$r0305-1048 (Linking)}, issn = {03051048}, journal = {Nucleic Acids Research}, number = {12}, pages = {1--9}, pmid = {19465396}, title = {{In silico selection of RNA aptamers}}, volume = {37}, year = {2009} } @article{Hu2015, author = {Hu, Wen-pin and Kumar, Jangam Vikram and Huang, Chun-jen and Chen, Wen-yih}, file = {:root/Downloads/658712.pdf:pdf}, title = {{Computational Selection of RNA Aptamer against Angiopoietin-2 and Experimental Evaluation}}, volume = {2015}, year = {2015} } @article{Luo2010, abstract = {It is well known that using random RNA/DNA sequences for SELEX experiments will generally yield low-complexity structures. Early experimental results suggest that having a structurally diverse library, which, for instance, includes high-order junctions, may prove useful in finding new functional motifs. Here, we develop two computational methods to generate sequences that exhibit higher structural complexity and can be used to increase the overall structural diversity of initial pools for in vitro selection experiments. Random Filtering selectively increases the number of five-way junctions in RNA/DNA pools, and Genetic Filtering designs RNA/DNA pools to a specified structure distribution, whether uniform or otherwise. We show that using our computationally designed DNA pool greatly improves access to highly complex sequence structures for SELEX experiments (without losing our ability to select for common one-way and two-way junction sequences).}, author = {Luo, Xuemei and McKeague, Maureen and Pitre, Sylvain and Dumontier, Michel and Green, James and Golshani, Ashkan and Derosa, Maria C and Dehne, Frank}, doi = {10.1261/rna.2102210}, file = {:root/Downloads/2252.pdf:pdf}, isbn = {1469-9001 (Electronic) 1355-8382 (Linking)}, issn = {1355-8382}, journal = {RNA (New York, N.Y.)}, keywords = {aptamer pool design,dna secondary structure,genetic algorithm,in vitro selection,random pool,rna}, number = {11}, pages = {2252--2262}, pmid = {20870801}, title = {{Computational approaches toward the design of pools for the in vitro selection of complex aptamers.}}, volume = {16}, year = {2010} } @article{Zhao2015, author = {Zhao, Zhen and Chen, Hongda and Ma, Lina and Liu, Dianjun and Wang, Zhenxin}, doi = {10.1039/C5AN00704F}, file = {:root/Downloads/c5an00704f.pdf:pdf}, issn = {0003-2654}, journal = {The Analyst}, number = {16}, pages = {5570--5577}, publisher = {Royal Society of Chemistry}, title = {{A label-free electrochemical impedance aptasensor for cylindrospermopsin detection based on thionine–graphene nanocomposites}}, url = {http://xlink.rsc.org/?DOI=C5AN00704F}, volume = {140}, } @article{Balbo2007, author = {Balbo, P. B. and Bohm, A.}, title = {Mechanism of poly(A) polymerase: structure of the enzyme-MgATP-RNA ternary complex and kinetic analysis}, journal = {Structure}, volume = {15}, number = {9}, pages = {1117-31}, note = {Balbo, Paul B Bohm, Andrew eng GM 065972/GM/NIGMS NIH HHS/ R01 GM065972/GM/NIGMS NIH HHS/ R01 GM065972-05/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural London, England : 1993 2007/09/14 09:00 Structure. 2007 Sep;15(9):1117-31.}, abstract = {We report the 1.8 A structure of yeast poly(A) polymerase (PAP) trapped in complex with ATP and a five residue poly(A) by mutation of the catalytically required aspartic acid 154 to alanine. The enzyme has undergone significant domain movement and reveals a closed conformation with extensive interactions between the substrates and all three polymerase domains. Both substrates and 31 buried water molecules are enclosed within a central cavity that is open at both ends. Four PAP mutants were subjected to detailed kinetic analysis, and studies of the adenylyltransfer (forward), pyrophosphorolysis (reverse), and nucleotidyltransfer reaction utilizing CTP for the mutants are presented. The results support a model in which binding of both poly(A) and the correct nucleotide, MgATP, induces a conformational change, resulting in formation of a stable, closed enzyme state. Thermodynamic considerations of the data are discussed as they pertain to domain closure, substrate specificity, and catalytic strategies utilized by PAP.}, keywords = {Adenosine Triphosphate/*metabolism Catalysis Kinetics Models, Molecular Mutagenesis, Site-Directed Polynucleotide Adenylyltransferase/chemistry/genetics/*metabolism Protein Conformation RNA/chemistry/*metabolism}, ISSN = {0969-2126 (Print) 0969-2126 (Linking)}, DOI = {10.1016/j.str.2007.07.010}, url = {http://www.ncbi.nlm.nih.gov/pubmed/17850751}, year = {2007}, type = {Journal Article} } @article{Baugh2000, author = {Baugh, C. and Grate, D. and Wilson, C.}, title = {2.8 angstrom crystal structure of the malachite green aptamer}, journal = {Journal of Molecular Biology}, volume = {301}, number = {1}, pages = {117-128}, note = {343MC Times Cited:90 Cited References Count:52}, abstract = {Previous in vitro selection experiments identified an RNA aptamer that recognizes the chromophore malachite green (MG) with a high level of affinity, and which undergoes site-specific cleavage following laser irradiation. To understand the mechanism by which this RNA folds to recognize specifically its ligand and the structural basis for chromophore-assisted laser inactivation, we have determined the 2.8 Angstrom crystal structure of the aptamer bound to tetramethylrosamine (TMR), a high-affinity MG analog. The ligand-binding site is defined by an asymmetric internal loop, flanked by a pair of helices. A U-turn and several non-canonical base interactions stabilize the folding of loop nucleotides around the TMR. The aptamer utilizes several tiers of stacked nucleotides arranged in pairs, triples, and a novel base quadruple to effectively encapsulate the ligand. Even in the absence of specific stabilizing hydrogen bonds, discrimination between related fluorophores and chromophores is possible due to tight packing in the RNA binding pocket, which severely limits the size and shape of recognized ligands. The site of laser-induced cleavage lies relatively far from the bound TMR (similar to 15 Angstrom). The unusual backbone conformation of the cleavage site nucleotide and its high level of solvent accessibility may, combine to allow preferential reaction with freely diffusing hydroxyl radicals generated at the bound ligand. Several observations, however, favor alternative mechanisms for cleavage, such as conformational changes in the aptamer or long-range electron transfer between the bound ligand and the cleavage site nucleotide. (C) 2000 Academic Press.}, keywords = {in vitro selection tetramethylrosamine base quadruple chromophore-assisted laser inactivation assisted laser inactivation ribosomal-rna binding rna DNA recognition resolution complex discrimination selection molecules}, ISSN = {0022-2836}, DOI = {10.1006/jmbi.2000.3951}, url = {://WOS:000088705300010}, year = {2000}, type = {Journal Article} } @article{Beckert2011, author = {Beckert, B. and Masquida, B.}, title = {Synthesis of RNA by in vitro transcription}, journal = {Methods Mol Biol}, volume = {703}, pages = {29-41}, note = {Beckert, Bertrand Masquida, Benoit eng Clifton, N.J. 2010/12/03 06:00 Methods Mol Biol. 2011;703:29-41. doi: 10.1007/978-1-59745-248-9_3.}, abstract = {In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases in mug to mg quantities. It is based on the engineering of a template that includes a bacteriophage promoter sequence (e.g. from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase. In vitro transcripts are used in analytical techniques (e.g. hybridization analysis), structural studies (for NMR and X-ray crystallography), in biochemical and genetic studies (e.g. as antisense reagents), and as functional molecules (ribozymes and aptamers).}, keywords = {Bacteriophage T7/genetics Base Sequence DNA-Directed RNA Polymerases/genetics In Vitro Techniques Molecular Biology/*methods Molecular Sequence Data Promoter Regions, Genetic/genetics RNA/*chemical synthesis *Transcription, Genetic}, ISSN = {1940-6029 (Electronic) 1064-3745 (Linking)}, DOI = {10.1007/978-1-59745-248-9_3}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21125481}, year = {2011}, type = {Journal Article} } @article{Bauxbaum2015, author = {Buxbaum, A. R. and Haimovich, G. and Singer, R. H.}, title = {In the right place at the right time: visualizing and understanding mRNA localization}, journal = {Nature Reviews Molecular Cell Biology}, volume = {16}, number = {2}, pages = {95-109}, note = {Ca1ad Times Cited:5 Cited References Count:176}, abstract = {The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization.}, keywords = {yeast saccharomyces-cerevisiae green fluorescent protein in-vitro reconstitution zipcode-binding protein actin gene-expression xenopus-oocytes hippocampal-neurons endoplasmic-reticulum dendritic transport particle tracking}, ISSN = {1471-0072}, DOI = {10.1038/nrm3918}, url = {://WOS:000348643800010}, year = {2015}, type = {Journal Article} } @article{Dean2014, author = {Dean, K. M. and Palmer, A. E.}, title = {Advances in fluorescence labeling strategies for dynamic cellular imaging}, journal = {Nature Chemical Biology}, volume = {10}, number = {7}, pages = {512-523}, note = {Aj7iu Times Cited:22 Cited References Count:96}, abstract = {Synergistic advances in optical physics, probe design, molecular biology, labeling techniques and computational analysis have propelled fluorescence imaging into new realms of spatiotemporal resolution and sensitivity. This review aims to discuss advances in fluorescent probes and live-cell labeling strategies, two areas that remain pivotal for future advances in imaging technology. Fluorescent protein- and bio-orthogonal-based methods for protein and RNA imaging are discussed as well as emerging bioengineering techniques that enable their expression at specific genomic loci (for example, CRISPR and TALENs). Important attributes that contribute to the success of each technique are emphasized, providing a guideline for future advances in dynamic live-cell imaging.}, keywords = {directed tosyl chemistry zinc-finger nucleases living cells in-vivo superresolution microscopy bacterial phytochrome stokes shift live cells protein rna}, ISSN = {1552-4450}, DOI = {10.1038/Nchembio.1556}, url = {://WOS:000337871200008}, year = {2014}, type = {Journal Article} } @article{Fernandey-Suarez2008, author = {Fernandez-Suarez, M. and Ting, A. Y.}, title = {Fluorescent probes for super-resolution imaging in living cells}, journal = {Nat Rev Mol Cell Biol}, volume = {9}, number = {12}, pages = {929-43}, note = {Fernandez-Suarez, Marta Ting, Alice Y eng Review England 2008/11/13 09:00 Nat Rev Mol Cell Biol. 2008 Dec;9(12):929-43. doi: 10.1038/nrm2531. Epub 2008 Nov 12.}, abstract = {In 1873, Ernst Abbe discovered that features closer than approximately 200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1-5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.}, keywords = {Animals Carbocyanines/metabolism Cells/*metabolism Coloring Agents/metabolism Diagnostic Imaging/*methods Fluorescent Dyes/*metabolism Green Fluorescent Proteins/metabolism Luminescent Agents/metabolism Microscopy, Fluorescence/*methods}, ISSN = {1471-0080 (Electronic) 1471-0072 (Linking)}, DOI = {10.1038/nrm2531}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19002208}, year = {2008}, type = {Journal Article} } @book{Hartmann2009 author = {Hartmann, Roland K.}, title = {Handbook of RNA biochemistry}, publisher = {Wiley-VCH}, address = {Weinheim}, edition = {1st student}, note = {2009499142 edited by Roland K. Hartmann ... [et al.]. ill. (some col.) ; 25 cm. Includes bibliographical references and index.}, keywords = {rna.}, pages = {xliii, 931 p.}, ISBN = {9783527325344 (pbk. alk. paper)}, year = {2009}, type = {Book} } @article{Höfer2013, author = {Höfer, K. and Langejürgen, L. V. and Jäschke, A.}, title = {Universal aptamer-based real-time monitoring of enzymatic RNA synthesis}, journal = {J Am Chem Soc}, volume = {135}, number = {37}, pages = {13692-4}, note = {Hofer, Katharina Langejurgen, Lisa V Jaschke, Andres eng 2013/09/03 06:00 J Am Chem Soc. 2013 Sep 18;135(37):13692-4. doi: 10.1021/ja407142f. Epub 2013 Sep 4.}, abstract = {In vitro transcription is an essential laboratory technique for enzymatic RNA synthesis. Unfortunately, no methods exist for analyzing quality and quantity of the synthesized RNA while the transcription proceeds. Here we describe a simple, robust, and universal system for monitoring and quantifying the synthesis of any RNA in real time without interference from abortive transcription byproducts. The distinguishing feature is a universal fluorescence module (UFM), consisting of the eGFP-like Spinach aptamer and a highly active hammerhead ribozyme, which is appended to the RNA of interest (ROI). In the transcription mixture, the primary transcript is cleaved rapidly behind the ROI, thereby releasing always the same UFM, independent of the ROI sequence, polymerase, or promoter used. The UFM binds to the target of the Spinach aptamer, the fluorogenic dye DFHBI, and thereby induces a strong fluorescence signal. This design allows real-time quantification, standardization, parallelization, and high-throughput screening.}, keywords = {Aptamers, Nucleotide/*chemical synthesis/chemistry *Biological Assay Exodeoxyribonucleases/*metabolism Fluorescence}, ISSN = {1520-5126 (Electronic) 0002-7863 (Linking)}, DOI = {10.1021/ja407142f}, url = {http://www.ncbi.nlm.nih.gov/pubmed/23991672}, year = {2013}, type = {Journal Article} } @article{Kellenberger2015, author = {Kellenberger, C. A. and Chen, C. and Whiteley, A. T. and Portnoy, D. A. and Hammond, M. C.}, title = {RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP}, journal = {Journal of the American Chemical Society}, volume = {137}, number = {20}, pages = {6432-6435}, note = {Cj3kv Times Cited:0 Cited References Count:24}, abstract = {Cyclic di-AMP (cdiA) is a second messenger predicted, to be widespread in Gram-positive bacteria, some Gram-negative bacteria, and Archaea. In the human pathogen Listeria monocytogenes, cdiA is an essential molecule that regulates metabolic function and cell wall homeostasis, and decreased levels of cdiA result in increased antibiotic susceptibility. We have generated fluorescent biosensors for cdiA through fusion of the Spinach2 aptamer to ligand-binding domains of cdiA riboswitches. The biosensor was used to visualize intra-cellular cdiA levels in live L. monocytogenes strains and to determine the catalytic domain of the phosphodiesterase PdeA. Furthermore; a flow cytometry assay based On this biosensor was used to screen for diadenylate cyclase activity and confirmed the enzymatic activity of DisA-like proteins from Clostridium difficile and Methanocaldococcus jannaschii. Thus, We have expanded the development of RNA-based biosensors for in vivo metabolite imaging in Gram-positive bacteria and have validated the first dinucleotide cyclase from Archaea.}, keywords = {bacterial 2nd-messenger ydao riboswitch small molecules nucleotides virulence reveals enters sense gmp}, ISSN = {0002-7863}, DOI = {10.1021/jacs.5b00275}, url = {://WOS:000355383500003}, year = {2015}, type = {Journal Article} } @article{Kellenberber2013, author = {Kellenberger, C. A. and Wilson, S. C. and Sales-Lee, J. and Hammond, M. C.}, title = {RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messengers Cyclic di-GMP and Cyclic AMP-GMP}, journal = {Journal of the American Chemical Society}, volume = {135}, number = {13}, pages = {4906-4909}, note = {121QM Times Cited:32 Cited References Count:24}, abstract = {Cyclic dinucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed RNA-based fluorescent biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-1 riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic dinucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.}, keywords = {escherichia-coli ligand-binding riboswitch diguanylate magnesium bacteria cgmp DNA}, ISSN = {0002-7863}, DOI = {10.1021/ja311960g}, url = {://WOS:000317259300002}, year = {2013}, type = {Journal Article} } @article{Paige2011, author = {Paige, J. S. and Wu, K. Y. and Jaffrey, S. R.}, title = {RNA mimics of green fluorescent protein}, journal = {Science}, volume = {333}, number = {6042}, pages = {642-6}, note = {Paige, Jeremy S Wu, Karen Y Jaffrey, Samie R eng NS064516/NS/NINDS NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ R01 NS064516-03/NS/NINDS NIH HHS/ T32 CA062948/CA/NCI NIH HHS/ T32 CA062948-14/CA/NCI NIH HHS/ T32CA062948/CA/NCI NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't New York, N.Y. 2011/07/30 06:00 Science. 2011 Jul 29;333(6042):642-6. doi: 10.1126/science.1207339.}, abstract = {Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here, we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex, termed Spinach, resembles enhanced GFP and emits a green fluorescence comparable in brightness with fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.}, keywords = {Aptamers, Nucleotide/*chemistry/*metabolism Benzyl Compounds/*chemistry/*metabolism Biomimetics Cell Nucleus/metabolism Cytoplasmic Granules/metabolism Cytosol/metabolism *Fluorescence Green Fluorescent Proteins/*chemistry HEK293 Cells Humans Imidazolines/*chemistry/*metabolism Molecular Mimicry Nucleic Acid Conformation Photobleaching Protein Binding RNA, Untranslated/metabolism SELEX Aptamer Technique Spectrometry, Fluorescence Sucrose/pharmacology}, ISSN = {1095-9203 (Electronic) 0036-8075 (Linking)}, DOI = {10.1126/science.1207339}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21798953}, year = {2011}, type = {Journal Article} } @article{Pothoulakis2014, author = {Pothoulakis, G. and Ceroni, F. and Reeve, B. and Ellis, T.}, title = {The spinach RNA aptamer as a characterization tool for synthetic biology}, journal = {ACS Synth Biol}, volume = {3}, number = {3}, pages = {182-7}, note = {Pothoulakis, Georgios Ceroni, Francesca Reeve, Benjamin Ellis, Tom eng Research Support, Non-U.S. Gov't 2013/09/03 06:00 ACS Synth Biol. 2014 Mar 21;3(3):182-7. doi: 10.1021/sb400089c. Epub 2013 Sep 13.}, abstract = {Characterization of genetic control elements is essential for the predictable engineering of synthetic biology systems. The current standard for in vivo characterization of control elements is through the use of fluorescent reporter proteins such as green fluorescent protein (GFP). Gene expression, however, involves not only protein production but also the production of mRNA. Here, we present the use of the Spinach aptamer sequence, an RNA mimic of GFP, as a tool to characterize mRNA expression in Escherichia coli. We show how the aptamer can be incorporated into gene expression cassettes and how co-expressing it with a red fluorescent protein (mRFP1) allows, for the first time, simultaneous measurement of mRNA and protein levels from engineered constructs. Using flow cytometry, we apply this tool here to evaluate ribosome binding site sequences and promoters and use it to highlight the differences in the temporal behavior of transcription and translation.}, keywords = {Aptamers, Nucleotide/*genetics/metabolism Binding Sites Escherichia coli/genetics/metabolism Genetic Engineering/*methods Luminescent Proteins/genetics/metabolism RNA/*genetics/metabolism RNA, Bacterial/genetics/metabolism Spinacia oleracea/*genetics Synthetic Biology/*methods}, ISSN = {2161-5063 (Electronic) 2161-5063 (Linking)}, DOI = {10.1021/sb400089c}, url = {http://www.ncbi.nlm.nih.gov/pubmed/23991760}, year = {2014}, type = {Journal Article} } @article{Sassanfar1993, author = {Sassanfar, M. and Szostak, J. W.}, title = {An Rna Motif That Binds Atp}, journal = {Nature}, volume = {364}, number = {6437}, pages = {550-553}, note = {Lq667 Times Cited:389 Cited References Count:17}, abstract = {RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules1,2. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.}, keywords = {molecular recognition receptor}, ISSN = {0028-0836}, DOI = {Doi 10.1038/364550a0}, url = {://WOS:A1993LQ66700062}, year = {1993}, type = {Journal Article} } @article{Sastry1997, author = {Sastry, S. S. and Ross, B. M.}, title = {Nuclease activity of T7 RNA polymerase and the heterogeneity of transcription elongation complexes}, journal = {J Biol Chem}, volume = {272}, number = {13}, pages = {8644-52}, note = {Sastry, S S Ross, B M eng Research Support, Non-U.S. Gov't 1997/03/28 J Biol Chem. 1997 Mar 28;272(13):8644-52.}, abstract = {We have discovered that T7 RNA polymerase, purified to apparent homogeneity from overexpressing Escherichia coli cells, possesses a DNase and an RNase activity. Mutations in the active center of T7 RNA polymerase abolished or greatly decreased the nuclease activity. This nuclease activity is specific for single-stranded DNA and RNA oligonucleotides and does not manifest on double-stranded DNAs. Under the conditions of promoter-driven transcription on double-stranded DNA, no nuclease activity was observed. The nuclease attacks DNA oligonucleotides in mono- or dinucleotide steps. The nuclease is a 3' to 5' exonuclease leaving a 3'-OH end, and it degrades DNA oligonucleotides to a minimum size of 3 to 5 nucleotides. It is completely dependent on Mg2+. The T7 RNA polymerase-nuclease is inhibited by T7 lysozyme and heparin, although not completely. In the presence of rNTPs, the nuclease activity is suppressed but an unusual 3'-end-initiated polymerase activity is unmasked. RNA from isolated pre-elongation and elongation complexes arrested by a psoralen roadblock or naturally paused at the 3'-end of an oligonucleotide template exhibited evidence of nuclease activity. The nuclease activity of T7 RNA polymerase is unrelated to pyrophosphorolysis. We propose that the nuclease of T7 RNA polymerase acts only in arrested or paused elongation complexes, and that in combination with the unusual 3'-end polymerizing activity, causes heterogeneity in elongation complexes. Additionally, during normal transcription elongation, the kinetic balance between nuclease and polymerase is shifted in favor of polymerase.}, keywords = {*Bacteriophage T7 Base Sequence DNA, Single-Stranded/metabolism DNA-Directed RNA Polymerases/*metabolism Deoxyribonucleases/metabolism Electrophoresis, Polyacrylamide Gel Enzyme Activation Furocoumarins/metabolism Heparin/pharmacology Magnesium/metabolism Molecular Sequence Data N-Acetylmuramoyl-L-alanine Amidase/metabolism *Peptide Elongation Factors RNA/metabolism Ribonucleases/metabolism Transcription, Genetic Viral Proteins}, ISSN = {0021-9258 (Print) 0021-9258 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/9079696}, year = {1997}, type = {Journal Article} } @article{Strack2013, author = {Strack, R. L. and Disney, M. D. and Jaffrey, S. R.}, title = {A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA}, journal = {Nat Methods}, volume = {10}, number = {12}, pages = {1219-24}, note = {Strack, Rita L Disney, Matthew D Jaffrey, Samie R eng F32 GM106683/GM/NIGMS NIH HHS/ GM079235/GM/NIGMS NIH HHS/ NS010249/NS/NINDS NIH HHS/ R01 EB010249/EB/NIBIB NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ Research Support, N.I.H., Extramural 2013/10/29 06:00 Nat Methods. 2013 Dec;10(12):1219-24. doi: 10.1038/nmeth.2701. Epub 2013 Oct 27.}, abstract = {Imaging RNA in living cells is a challenging problem in cell biology. One strategy for genetically encoding fluorescent RNAs is to express them as fusions with Spinach, an 'RNA mimic of GFP'. We found that Spinach was dimmer than expected when used to tag constructs in living cells owing to a combination of thermal instability and a propensity for misfolding. Using systematic mutagenesis, we generated Spinach2 that overcomes these issues and can be used to image diverse RNAs. Using Spinach2, we detailed the dynamics of the CGG trinucleotide repeat-containing 'toxic RNA' associated with Fragile X-associated tremor/ataxia syndrome, and show that these RNAs form nuclear foci with unexpected morphological plasticity that is regulated by the cell cycle and by small molecules. Together, these data demonstrate that Spinach2 exhibits improved versatility for fluorescently labeling RNAs in living cells.}, keywords = {Animals Base Sequence COS Cells Cercopithecus aethiops DNA Mutational Analysis Escherichia coli/metabolism Fluorescent Dyes/chemistry Green Fluorescent Proteins/metabolism HEK293 Cells Humans Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Protein Denaturation Protein Folding RNA/*chemistry RNA, Untranslated/*chemistry Temperature *Trinucleotide Repeats}, ISSN = {1548-7105 (Electronic) 1548-7091 (Linking)}, DOI = {10.1038/nmeth.2701}, url = {http://www.ncbi.nlm.nih.gov/pubmed/24162923}, year = {2013}, type = {Journal Article} } @article{Strack2015, author = {Strack, R. L. and Jaffrey, S. R.}, title = {Live-cell imaging of mammalian RNAs with Spinach2}, journal = {Methods Enzymol}, volume = {550}, pages = {129-46}, note = {Strack, Rita L Jaffrey, Samie R eng F32 GM106683/GM/NIGMS NIH HHS/ NS010249/NS/NINDS NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ Research Support, N.I.H., Extramural 2015/01/22 06:00 Methods Enzymol. 2015;550:129-46. doi: 10.1016/bs.mie.2014.10.044. Epub 2015 Jan 6.}, abstract = {The ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of imaging individual RNAs of interest in living cells, each with their own advantages and limitations. This chapter provides an overview of current methods of live-cell RNA imaging, including a detailed discussion of genetically encoded strategies for labeling RNAs in mammalian cells. This chapter then focuses on the development and use of "RNA mimics of GFP" or Spinach technology for tagging mammalian RNAs and includes a detailed protocol for imaging 5S and CGG60 RNA with the recently described Spinach2 tag.}, ISSN = {1557-7988 (Electronic) 0076-6879 (Linking)}, DOI = {10.1016/bs.mie.2014.10.044}, url = {http://www.ncbi.nlm.nih.gov/pubmed/25605384}, year = {2015}, type = {Journal Article} } @article{Tyagi2009, author = {Tyagi, S.}, title = {Imaging intracellular RNA distribution and dynamics in living cells}, journal = {Nat Methods}, volume = {6}, number = {5}, pages = {331-8}, note = {Tyagi, Sanjay eng MH079197/MH/NIMH NIH HHS/ Research Support, N.I.H., Extramural Review 2009/05/01 09:00 Nat Methods. 2009 May;6(5):331-8. doi: 10.1038/nmeth.1321.}, abstract = {Powerful methods now allow the imaging of specific mRNAs in living cells. These methods enlist fluorescent proteins to illuminate mRNAs, use labeled oligonucleotide probes and exploit aptamers that render organic dyes fluorescent. The intracellular dynamics of mRNA synthesis, transport and localization can be analyzed at higher temporal resolution with these methods than has been possible with traditional fixed-cell or biochemical approaches. These methods have also been adopted to visualize and track single mRNA molecules in real time. This review explores the promises and limitations of these methods.}, keywords = {Aptamers, Nucleotide/chemistry Cell Survival Cells/*cytology/*metabolism Cytophotometry/*methods Fluorescent Dyes/chemistry Green Fluorescent Proteins/chemistry/genetics Microscopy, Fluorescence/methods Oligonucleotide Probes/chemistry RNA, Messenger/*analysis/chemistry/*metabolism RNA-Binding Proteins/chemistry/genetics Recombinant Fusion Proteins/chemistry/genetics}, ISSN = {1548-7105 (Electronic) 1548-7091 (Linking)}, DOI = {10.1038/nmeth.1321}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19404252}, year = {2009}, type = {Journal Article} } e@article{Balbo2007, author = {Balbo, P. B. and Bohm, A.}, title = {Mechanism of poly(A) polymerase: structure of the enzyme-MgATP-RNA ternary complex and kinetic analysis}, journal = {Structure}, volume = {15}, number = {9}, pages = {1117-31}, note = {Balbo, Paul B Bohm, Andrew eng GM 065972/GM/NIGMS NIH HHS/ R01 GM065972/GM/NIGMS NIH HHS/ R01 GM065972-05/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural London, England : 1993 2007/09/14 09:00 Structure. 2007 Sep;15(9):1117-31.}, abstract = {We report the 1.8 A structure of yeast poly(A) polymerase (PAP) trapped in complex with ATP and a five residue poly(A) by mutation of the catalytically required aspartic acid 154 to alanine. The enzyme has undergone significant domain movement and reveals a closed conformation with extensive interactions between the substrates and all three polymerase domains. Both substrates and 31 buried water molecules are enclosed within a central cavity that is open at both ends. Four PAP mutants were subjected to detailed kinetic analysis, and studies of the adenylyltransfer (forward), pyrophosphorolysis (reverse), and nucleotidyltransfer reaction utilizing CTP for the mutants are presented. The results support a model in which binding of both poly(A) and the correct nucleotide, MgATP, induces a conformational change, resulting in formation of a stable, closed enzyme state. Thermodynamic considerations of the data are discussed as they pertain to domain closure, substrate specificity, and catalytic strategies utilized by PAP.}, keywords = {Adenosine Triphosphate/*metabolism Catalysis Kinetics Models, Molecular Mutagenesis, Site-Directed Polynucleotide Adenylyltransferase/chemistry/genetics/*metabolism Protein Conformation RNA/chemistry/*metabolism}, ISSN = {0969-2126 (Print) 0969-2126 (Linking)}, DOI = {10.1016/j.str.2007.07.010}, url = {http://www.ncbi.nlm.nih.gov/pubmed/17850751}, year = {2007}, type = {Journal Article} } @article{Baugh2000, author = {Baugh, C. and Grate, D. and Wilson, C.}, title = {2.8 angstrom crystal structure of the malachite green aptamer}, journal = {Journal of Molecular Biology}, volume = {301}, number = {1}, pages = {117-128}, note = {343MC Times Cited:90 Cited References Count:52}, abstract = {Previous in vitro selection experiments identified an RNA aptamer that recognizes the chromophore malachite green (MG) with a high level of affinity, and which undergoes site-specific cleavage following laser irradiation. To understand the mechanism by which this RNA folds to recognize specifically its ligand and the structural basis for chromophore-assisted laser inactivation, we have determined the 2.8 Angstrom crystal structure of the aptamer bound to tetramethylrosamine (TMR), a high-affinity MG analog. The ligand-binding site is defined by an asymmetric internal loop, flanked by a pair of helices. A U-turn and several non-canonical base interactions stabilize the folding of loop nucleotides around the TMR. The aptamer utilizes several tiers of stacked nucleotides arranged in pairs, triples, and a novel base quadruple to effectively encapsulate the ligand. Even in the absence of specific stabilizing hydrogen bonds, discrimination between related fluorophores and chromophores is possible due to tight packing in the RNA binding pocket, which severely limits the size and shape of recognized ligands. The site of laser-induced cleavage lies relatively far from the bound TMR (similar to 15 Angstrom). The unusual backbone conformation of the cleavage site nucleotide and its high level of solvent accessibility may, combine to allow preferential reaction with freely diffusing hydroxyl radicals generated at the bound ligand. Several observations, however, favor alternative mechanisms for cleavage, such as conformational changes in the aptamer or long-range electron transfer between the bound ligand and the cleavage site nucleotide. (C) 2000 Academic Press.}, keywords = {in vitro selection tetramethylrosamine base quadruple chromophore-assisted laser inactivation assisted laser inactivation ribosomal-rna binding rna DNA recognition resolution complex discrimination selection molecules}, ISSN = {0022-2836}, DOI = {10.1006/jmbi.2000.3951}, url = {://WOS:000088705300010}, year = {2000}, type = {Journal Article} } @article{Beckert2011, author = {Beckert, B. and Masquida, B.}, title = {Synthesis of RNA by in vitro transcription}, journal = {Methods Mol Biol}, volume = {703}, pages = {29-41}, note = {Beckert, Bertrand Masquida, Benoit eng Clifton, N.J. 2010/12/03 06:00 Methods Mol Biol. 2011;703:29-41. doi: 10.1007/978-1-59745-248-9_3.}, abstract = {In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases in mug to mg quantities. It is based on the engineering of a template that includes a bacteriophage promoter sequence (e.g. from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase. In vitro transcripts are used in analytical techniques (e.g. hybridization analysis), structural studies (for NMR and X-ray crystallography), in biochemical and genetic studies (e.g. as antisense reagents), and as functional molecules (ribozymes and aptamers).}, keywords = {Bacteriophage T7/genetics Base Sequence DNA-Directed RNA Polymerases/genetics In Vitro Techniques Molecular Biology/*methods Molecular Sequence Data Promoter Regions, Genetic/genetics RNA/*chemical synthesis *Transcription, Genetic}, ISSN = {1940-6029 (Electronic) 1064-3745 (Linking)}, DOI = {10.1007/978-1-59745-248-9_3}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21125481}, year = {2011}, type = {Journal Article} } @article{Bauxbaum2015, author = {Buxbaum, A. R. and Haimovich, G. and Singer, R. H.}, title = {In the right place at the right time: visualizing and understanding mRNA localization}, journal = {Nature Reviews Molecular Cell Biology}, volume = {16}, number = {2}, pages = {95-109}, note = {Ca1ad Times Cited:5 Cited References Count:176}, abstract = {The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization.}, keywords = {yeast saccharomyces-cerevisiae green fluorescent protein in-vitro reconstitution zipcode-binding protein actin gene-expression xenopus-oocytes hippocampal-neurons endoplasmic-reticulum dendritic transport particle tracking}, ISSN = {1471-0072}, DOI = {10.1038/nrm3918}, url = {://WOS:000348643800010}, year = {2015}, type = {Journal Article} } @article{Dean2014, author = {Dean, K. M. and Palmer, A. E.}, title = {Advances in fluorescence labeling strategies for dynamic cellular imaging}, journal = {Nature Chemical Biology}, volume = {10}, number = {7}, pages = {512-523}, note = {Aj7iu Times Cited:22 Cited References Count:96}, abstract = {Synergistic advances in optical physics, probe design, molecular biology, labeling techniques and computational analysis have propelled fluorescence imaging into new realms of spatiotemporal resolution and sensitivity. This review aims to discuss advances in fluorescent probes and live-cell labeling strategies, two areas that remain pivotal for future advances in imaging technology. Fluorescent protein- and bio-orthogonal-based methods for protein and RNA imaging are discussed as well as emerging bioengineering techniques that enable their expression at specific genomic loci (for example, CRISPR and TALENs). Important attributes that contribute to the success of each technique are emphasized, providing a guideline for future advances in dynamic live-cell imaging.}, keywords = {directed tosyl chemistry zinc-finger nucleases living cells in-vivo superresolution microscopy bacterial phytochrome stokes shift live cells protein rna}, ISSN = {1552-4450}, DOI = {10.1038/Nchembio.1556}, url = {://WOS:000337871200008}, year = {2014}, type = {Journal Article} } @article{Fernandey-Suarez2008, author = {Fernandez-Suarez, M. and Ting, A. Y.}, title = {Fluorescent probes for super-resolution imaging in living cells}, journal = {Nat Rev Mol Cell Biol}, volume = {9}, number = {12}, pages = {929-43}, note = {Fernandez-Suarez, Marta Ting, Alice Y eng Review England 2008/11/13 09:00 Nat Rev Mol Cell Biol. 2008 Dec;9(12):929-43. doi: 10.1038/nrm2531. Epub 2008 Nov 12.}, abstract = {In 1873, Ernst Abbe discovered that features closer than approximately 200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1-5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.}, keywords = {Animals Carbocyanines/metabolism Cells/*metabolism Coloring Agents/metabolism Diagnostic Imaging/*methods Fluorescent Dyes/*metabolism Green Fluorescent Proteins/metabolism Luminescent Agents/metabolism Microscopy, Fluorescence/*methods}, ISSN = {1471-0080 (Electronic) 1471-0072 (Linking)}, DOI = {10.1038/nrm2531}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19002208}, year = {2008}, type = {Journal Article} } @book{Hartmann2009 author = {Hartmann, Roland K.}, title = {Handbook of RNA biochemistry}, publisher = {Wiley-VCH}, address = {Weinheim}, edition = {1st student}, note = {2009499142 edited by Roland K. Hartmann ... [et al.]. ill. (some col.) ; 25 cm. Includes bibliographical references and index.}, keywords = {rna.}, pages = {xliii, 931 p.}, ISBN = {9783527325344 (pbk. alk. paper)}, year = {2009}, type = {Book} } @article{Höfer2013, author = {Höfer, K. and Langejürgen, L. V. and Jäschke, A.}, title = {Universal aptamer-based real-time monitoring of enzymatic RNA synthesis}, journal = {J Am Chem Soc}, volume = {135}, number = {37}, pages = {13692-4}, note = {Hofer, Katharina Langejurgen, Lisa V Jaschke, Andres eng 2013/09/03 06:00 J Am Chem Soc. 2013 Sep 18;135(37):13692-4. doi: 10.1021/ja407142f. Epub 2013 Sep 4.}, abstract = {In vitro transcription is an essential laboratory technique for enzymatic RNA synthesis. Unfortunately, no methods exist for analyzing quality and quantity of the synthesized RNA while the transcription proceeds. Here we describe a simple, robust, and universal system for monitoring and quantifying the synthesis of any RNA in real time without interference from abortive transcription byproducts. The distinguishing feature is a universal fluorescence module (UFM), consisting of the eGFP-like Spinach aptamer and a highly active hammerhead ribozyme, which is appended to the RNA of interest (ROI). In the transcription mixture, the primary transcript is cleaved rapidly behind the ROI, thereby releasing always the same UFM, independent of the ROI sequence, polymerase, or promoter used. The UFM binds to the target of the Spinach aptamer, the fluorogenic dye DFHBI, and thereby induces a strong fluorescence signal. This design allows real-time quantification, standardization, parallelization, and high-throughput screening.}, keywords = {Aptamers, Nucleotide/*chemical synthesis/chemistry *Biological Assay Exodeoxyribonucleases/*metabolism Fluorescence}, ISSN = {1520-5126 (Electronic) 0002-7863 (Linking)}, DOI = {10.1021/ja407142f}, url = {http://www.ncbi.nlm.nih.gov/pubmed/23991672}, year = {2013}, type = {Journal Article} } @article{Kellenberger2015, author = {Kellenberger, C. A. and Chen, C. and Whiteley, A. T. and Portnoy, D. A. and Hammond, M. C.}, title = {RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP}, journal = {Journal of the American Chemical Society}, volume = {137}, number = {20}, pages = {6432-6435}, note = {Cj3kv Times Cited:0 Cited References Count:24}, abstract = {Cyclic di-AMP (cdiA) is a second messenger predicted, to be widespread in Gram-positive bacteria, some Gram-negative bacteria, and Archaea. In the human pathogen Listeria monocytogenes, cdiA is an essential molecule that regulates metabolic function and cell wall homeostasis, and decreased levels of cdiA result in increased antibiotic susceptibility. We have generated fluorescent biosensors for cdiA through fusion of the Spinach2 aptamer to ligand-binding domains of cdiA riboswitches. The biosensor was used to visualize intra-cellular cdiA levels in live L. monocytogenes strains and to determine the catalytic domain of the phosphodiesterase PdeA. Furthermore; a flow cytometry assay based On this biosensor was used to screen for diadenylate cyclase activity and confirmed the enzymatic activity of DisA-like proteins from Clostridium difficile and Methanocaldococcus jannaschii. Thus, We have expanded the development of RNA-based biosensors for in vivo metabolite imaging in Gram-positive bacteria and have validated the first dinucleotide cyclase from Archaea.}, keywords = {bacterial 2nd-messenger ydao riboswitch small molecules nucleotides virulence reveals enters sense gmp}, ISSN = {0002-7863}, DOI = {10.1021/jacs.5b00275}, url = {://WOS:000355383500003}, year = {2015}, type = {Journal Article} } @article{Kellenberber2013, author = {Kellenberger, C. A. and Wilson, S. C. and Sales-Lee, J. and Hammond, M. C.}, title = {RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messengers Cyclic di-GMP and Cyclic AMP-GMP}, journal = {Journal of the American Chemical Society}, volume = {135}, number = {13}, pages = {4906-4909}, note = {121QM Times Cited:32 Cited References Count:24}, abstract = {Cyclic dinucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed RNA-based fluorescent biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-1 riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic dinucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.}, keywords = {escherichia-coli ligand-binding riboswitch diguanylate magnesium bacteria cgmp DNA}, ISSN = {0002-7863}, DOI = {10.1021/ja311960g}, url = {://WOS:000317259300002}, year = {2013}, type = {Journal Article} } @article{Paige2011, author = {Paige, J. S. and Wu, K. Y. and Jaffrey, S. R.}, title = {RNA mimics of green fluorescent protein}, journal = {Science}, volume = {333}, number = {6042}, pages = {642-6}, note = {Paige, Jeremy S Wu, Karen Y Jaffrey, Samie R eng NS064516/NS/NINDS NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ R01 NS064516-03/NS/NINDS NIH HHS/ T32 CA062948/CA/NCI NIH HHS/ T32 CA062948-14/CA/NCI NIH HHS/ T32CA062948/CA/NCI NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't New York, N.Y. 2011/07/30 06:00 Science. 2011 Jul 29;333(6042):642-6. doi: 10.1126/science.1207339.}, abstract = {Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here, we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex, termed Spinach, resembles enhanced GFP and emits a green fluorescence comparable in brightness with fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.}, keywords = {Aptamers, Nucleotide/*chemistry/*metabolism Benzyl Compounds/*chemistry/*metabolism Biomimetics Cell Nucleus/metabolism Cytoplasmic Granules/metabolism Cytosol/metabolism *Fluorescence Green Fluorescent Proteins/*chemistry HEK293 Cells Humans Imidazolines/*chemistry/*metabolism Molecular Mimicry Nucleic Acid Conformation Photobleaching Protein Binding RNA, Untranslated/metabolism SELEX Aptamer Technique Spectrometry, Fluorescence Sucrose/pharmacology}, ISSN = {1095-9203 (Electronic) 0036-8075 (Linking)}, DOI = {10.1126/science.1207339}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21798953}, year = {2011}, type = {Journal Article} } @article{Pothoulakis2014, author = {Pothoulakis, G. and Ceroni, F. and Reeve, B. and Ellis, T.}, title = {The spinach RNA aptamer as a characterization tool for synthetic biology}, journal = {ACS Synth Biol}, volume = {3}, number = {3}, pages = {182-7}, note = {Pothoulakis, Georgios Ceroni, Francesca Reeve, Benjamin Ellis, Tom eng Research Support, Non-U.S. Gov't 2013/09/03 06:00 ACS Synth Biol. 2014 Mar 21;3(3):182-7. doi: 10.1021/sb400089c. Epub 2013 Sep 13.}, abstract = {Characterization of genetic control elements is essential for the predictable engineering of synthetic biology systems. The current standard for in vivo characterization of control elements is through the use of fluorescent reporter proteins such as green fluorescent protein (GFP). Gene expression, however, involves not only protein production but also the production of mRNA. Here, we present the use of the Spinach aptamer sequence, an RNA mimic of GFP, as a tool to characterize mRNA expression in Escherichia coli. We show how the aptamer can be incorporated into gene expression cassettes and how co-expressing it with a red fluorescent protein (mRFP1) allows, for the first time, simultaneous measurement of mRNA and protein levels from engineered constructs. Using flow cytometry, we apply this tool here to evaluate ribosome binding site sequences and promoters and use it to highlight the differences in the temporal behavior of transcription and translation.}, keywords = {Aptamers, Nucleotide/*genetics/metabolism Binding Sites Escherichia coli/genetics/metabolism Genetic Engineering/*methods Luminescent Proteins/genetics/metabolism RNA/*genetics/metabolism RNA, Bacterial/genetics/metabolism Spinacia oleracea/*genetics Synthetic Biology/*methods}, ISSN = {2161-5063 (Electronic) 2161-5063 (Linking)}, DOI = {10.1021/sb400089c}, url = {http://www.ncbi.nlm.nih.gov/pubmed/23991760}, year = {2014}, type = {Journal Article} } @article{Sassanfar1993, author = {Sassanfar, M. and Szostak, J. W.}, title = {An Rna Motif That Binds Atp}, journal = {Nature}, volume = {364}, number = {6437}, pages = {550-553}, note = {Lq667 Times Cited:389 Cited References Count:17}, abstract = {RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules1,2. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.}, keywords = {molecular recognition receptor}, ISSN = {0028-0836}, DOI = {Doi 10.1038/364550a0}, url = {://WOS:A1993LQ66700062}, year = {1993}, type = {Journal Article} } @article{Sastry1997, author = {Sastry, S. S. and Ross, B. M.}, title = {Nuclease activity of T7 RNA polymerase and the heterogeneity of transcription elongation complexes}, journal = {J Biol Chem}, volume = {272}, number = {13}, pages = {8644-52}, note = {Sastry, S S Ross, B M eng Research Support, Non-U.S. Gov't 1997/03/28 J Biol Chem. 1997 Mar 28;272(13):8644-52.}, abstract = {We have discovered that T7 RNA polymerase, purified to apparent homogeneity from overexpressing Escherichia coli cells, possesses a DNase and an RNase activity. Mutations in the active center of T7 RNA polymerase abolished or greatly decreased the nuclease activity. This nuclease activity is specific for single-stranded DNA and RNA oligonucleotides and does not manifest on double-stranded DNAs. Under the conditions of promoter-driven transcription on double-stranded DNA, no nuclease activity was observed. The nuclease attacks DNA oligonucleotides in mono- or dinucleotide steps. The nuclease is a 3' to 5' exonuclease leaving a 3'-OH end, and it degrades DNA oligonucleotides to a minimum size of 3 to 5 nucleotides. It is completely dependent on Mg2+. The T7 RNA polymerase-nuclease is inhibited by T7 lysozyme and heparin, although not completely. In the presence of rNTPs, the nuclease activity is suppressed but an unusual 3'-end-initiated polymerase activity is unmasked. RNA from isolated pre-elongation and elongation complexes arrested by a psoralen roadblock or naturally paused at the 3'-end of an oligonucleotide template exhibited evidence of nuclease activity. The nuclease activity of T7 RNA polymerase is unrelated to pyrophosphorolysis. We propose that the nuclease of T7 RNA polymerase acts only in arrested or paused elongation complexes, and that in combination with the unusual 3'-end polymerizing activity, causes heterogeneity in elongation complexes. Additionally, during normal transcription elongation, the kinetic balance between nuclease and polymerase is shifted in favor of polymerase.}, keywords = {*Bacteriophage T7 Base Sequence DNA, Single-Stranded/metabolism DNA-Directed RNA Polymerases/*metabolism Deoxyribonucleases/metabolism Electrophoresis, Polyacrylamide Gel Enzyme Activation Furocoumarins/metabolism Heparin/pharmacology Magnesium/metabolism Molecular Sequence Data N-Acetylmuramoyl-L-alanine Amidase/metabolism *Peptide Elongation Factors RNA/metabolism Ribonucleases/metabolism Transcription, Genetic Viral Proteins}, ISSN = {0021-9258 (Print) 0021-9258 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/9079696}, year = {1997}, type = {Journal Article} } @article{Strack2013, author = {Strack, R. L. and Disney, M. D. and Jaffrey, S. R.}, title = {A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA}, journal = {Nat Methods}, volume = {10}, number = {12}, pages = {1219-24}, note = {Strack, Rita L Disney, Matthew D Jaffrey, Samie R eng F32 GM106683/GM/NIGMS NIH HHS/ GM079235/GM/NIGMS NIH HHS/ NS010249/NS/NINDS NIH HHS/ R01 EB010249/EB/NIBIB NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ Research Support, N.I.H., Extramural 2013/10/29 06:00 Nat Methods. 2013 Dec;10(12):1219-24. doi: 10.1038/nmeth.2701. Epub 2013 Oct 27.}, abstract = {Imaging RNA in living cells is a challenging problem in cell biology. One strategy for genetically encoding fluorescent RNAs is to express them as fusions with Spinach, an 'RNA mimic of GFP'. We found that Spinach was dimmer than expected when used to tag constructs in living cells owing to a combination of thermal instability and a propensity for misfolding. Using systematic mutagenesis, we generated Spinach2 that overcomes these issues and can be used to image diverse RNAs. Using Spinach2, we detailed the dynamics of the CGG trinucleotide repeat-containing 'toxic RNA' associated with Fragile X-associated tremor/ataxia syndrome, and show that these RNAs form nuclear foci with unexpected morphological plasticity that is regulated by the cell cycle and by small molecules. Together, these data demonstrate that Spinach2 exhibits improved versatility for fluorescently labeling RNAs in living cells.}, keywords = {Animals Base Sequence COS Cells Cercopithecus aethiops DNA Mutational Analysis Escherichia coli/metabolism Fluorescent Dyes/chemistry Green Fluorescent Proteins/metabolism HEK293 Cells Humans Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Protein Denaturation Protein Folding RNA/*chemistry RNA, Untranslated/*chemistry Temperature *Trinucleotide Repeats}, ISSN = {1548-7105 (Electronic) 1548-7091 (Linking)}, DOI = {10.1038/nmeth.2701}, url = {http://www.ncbi.nlm.nih.gov/pubmed/24162923}, year = {2013}, type = {Journal Article} } @article{Strack2015, author = {Strack, R. L. and Jaffrey, S. R.}, title = {Live-cell imaging of mammalian RNAs with Spinach2}, journal = {Methods Enzymol}, volume = {550}, pages = {129-46}, note = {Strack, Rita L Jaffrey, Samie R eng F32 GM106683/GM/NIGMS NIH HHS/ NS010249/NS/NINDS NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ Research Support, N.I.H., Extramural 2015/01/22 06:00 Methods Enzymol. 2015;550:129-46. doi: 10.1016/bs.mie.2014.10.044. Epub 2015 Jan 6.}, abstract = {The ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of imaging individual RNAs of interest in living cells, each with their own advantages and limitations. This chapter provides an overview of current methods of live-cell RNA imaging, including a detailed discussion of genetically encoded strategies for labeling RNAs in mammalian cells. This chapter then focuses on the development and use of "RNA mimics of GFP" or Spinach technology for tagging mammalian RNAs and includes a detailed protocol for imaging 5S and CGG60 RNA with the recently described Spinach2 tag.}, ISSN = {1557-7988 (Electronic) 0076-6879 (Linking)}, DOI = {10.1016/bs.mie.2014.10.044}, url = {http://www.ncbi.nlm.nih.gov/pubmed/25605384}, year = {2015}, type = {Journal Article} } @article{Tyagi2009, author = {Tyagi, S.}, title = {Imaging intracellular RNA distribution and dynamics in living cells}, journal = {Nat Methods}, volume = {6}, number = {5}, pages = {331-8}, note = {Tyagi, Sanjay eng MH079197/MH/NIMH NIH HHS/ Research Support, N.I.H., Extramural Review 2009/05/01 09:00 Nat Methods. 2009 May;6(5):331-8. doi: 10.1038/nmeth.1321.}, abstract = {Powerful methods now allow the imaging of specific mRNAs in living cells. These methods enlist fluorescent proteins to illuminate mRNAs, use labeled oligonucleotide probes and exploit aptamers that render organic dyes fluorescent. The intracellular dynamics of mRNA synthesis, transport and localization can be analyzed at higher temporal resolution with these methods than has been possible with traditional fixed-cell or biochemical approaches. These methods have also been adopted to visualize and track single mRNA molecules in real time. This review explores the promises and limitations of these methods.}, keywords = {Aptamers, Nucleotide/chemistry Cell Survival Cells/*cytology/*metabolism Cytophotometry/*methods Fluorescent Dyes/chemistry Green Fluorescent Proteins/chemistry/genetics Microscopy, Fluorescence/methods Oligonucleotide Probes/chemistry RNA, Messenger/*analysis/chemistry/*metabolism RNA-Binding Proteins/chemistry/genetics Recombinant Fusion Proteins/chemistry/genetics}, ISSN = {1548-7105 (Electronic) 1548-7091 (Linking)}, DOI = {10.1038/nmeth.1321}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19404252}, year = {2009}, type = {Journal Article} } {Balbo2007, author = {Balbo, P. B. and Bohm, A.}, title = {Mechanism of poly(A) polymerase: structure of the enzyme-MgATP-RNA ternary complex and kinetic analysis}, journal = {Structure}, volume = {15}, number = {9}, pages = {1117-31}, note = {Balbo, Paul B Bohm, Andrew eng GM 065972/GM/NIGMS NIH HHS/ R01 GM065972/GM/NIGMS NIH HHS/ R01 GM065972-05/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural London, England : 1993 2007/09/14 09:00 Structure. 2007 Sep;15(9):1117-31.}, abstract = {We report the 1.8 A structure of yeast poly(A) polymerase (PAP) trapped in complex with ATP and a five residue poly(A) by mutation of the catalytically required aspartic acid 154 to alanine. The enzyme has undergone significant domain movement and reveals a closed conformation with extensive interactions between the substrates and all three polymerase domains. Both substrates and 31 buried water molecules are enclosed within a central cavity that is open at both ends. Four PAP mutants were subjected to detailed kinetic analysis, and studies of the adenylyltransfer (forward), pyrophosphorolysis (reverse), and nucleotidyltransfer reaction utilizing CTP for the mutants are presented. The results support a model in which binding of both poly(A) and the correct nucleotide, MgATP, induces a conformational change, resulting in formation of a stable, closed enzyme state. Thermodynamic considerations of the data are discussed as they pertain to domain closure, substrate specificity, and catalytic strategies utilized by PAP.}, keywords = {Adenosine Triphosphate/*metabolism Catalysis Kinetics Models, Molecular Mutagenesis, Site-Directed Polynucleotide Adenylyltransferase/chemistry/genetics/*metabolism Protein Conformation RNA/chemistry/*metabolism}, ISSN = {0969-2126 (Print) 0969-2126 (Linking)}, DOI = {10.1016/j.str.2007.07.010}, url = {http://www.ncbi.nlm.nih.gov/pubmed/17850751}, year = {2007}, type = {Journal Article} } @article{Baugh2000, author = {Baugh, C. and Grate, D. and Wilson, C.}, title = {2.8 angstrom crystal structure of the malachite green aptamer}, journal = {Journal of Molecular Biology}, volume = {301}, number = {1}, pages = {117-128}, note = {343MC Times Cited:90 Cited References Count:52}, abstract = {Previous in vitro selection experiments identified an RNA aptamer that recognizes the chromophore malachite green (MG) with a high level of affinity, and which undergoes site-specific cleavage following laser irradiation. To understand the mechanism by which this RNA folds to recognize specifically its ligand and the structural basis for chromophore-assisted laser inactivation, we have determined the 2.8 Angstrom crystal structure of the aptamer bound to tetramethylrosamine (TMR), a high-affinity MG analog. The ligand-binding site is defined by an asymmetric internal loop, flanked by a pair of helices. A U-turn and several non-canonical base interactions stabilize the folding of loop nucleotides around the TMR. The aptamer utilizes several tiers of stacked nucleotides arranged in pairs, triples, and a novel base quadruple to effectively encapsulate the ligand. Even in the absence of specific stabilizing hydrogen bonds, discrimination between related fluorophores and chromophores is possible due to tight packing in the RNA binding pocket, which severely limits the size and shape of recognized ligands. The site of laser-induced cleavage lies relatively far from the bound TMR (similar to 15 Angstrom). The unusual backbone conformation of the cleavage site nucleotide and its high level of solvent accessibility may, combine to allow preferential reaction with freely diffusing hydroxyl radicals generated at the bound ligand. Several observations, however, favor alternative mechanisms for cleavage, such as conformational changes in the aptamer or long-range electron transfer between the bound ligand and the cleavage site nucleotide. (C) 2000 Academic Press.}, keywords = {in vitro selection tetramethylrosamine base quadruple chromophore-assisted laser inactivation assisted laser inactivation ribosomal-rna binding rna DNA recognition resolution complex discrimination selection molecules}, ISSN = {0022-2836}, DOI = {10.1006/jmbi.2000.3951}, url = {://WOS:000088705300010}, year = {2000}, type = {Journal Article} } @article{Beckert2011, author = {Beckert, B. and Masquida, B.}, title = {Synthesis of RNA by in vitro transcription}, journal = {Methods Mol Biol}, volume = {703}, pages = {29-41}, note = {Beckert, Bertrand Masquida, Benoit eng Clifton, N.J. 2010/12/03 06:00 Methods Mol Biol. 2011;703:29-41. doi: 10.1007/978-1-59745-248-9_3.}, abstract = {In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases in mug to mg quantities. It is based on the engineering of a template that includes a bacteriophage promoter sequence (e.g. from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase. In vitro transcripts are used in analytical techniques (e.g. hybridization analysis), structural studies (for NMR and X-ray crystallography), in biochemical and genetic studies (e.g. as antisense reagents), and as functional molecules (ribozymes and aptamers).}, keywords = {Bacteriophage T7/genetics Base Sequence DNA-Directed RNA Polymerases/genetics In Vitro Techniques Molecular Biology/*methods Molecular Sequence Data Promoter Regions, Genetic/genetics RNA/*chemical synthesis *Transcription, Genetic}, ISSN = {1940-6029 (Electronic) 1064-3745 (Linking)}, DOI = {10.1007/978-1-59745-248-9_3}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21125481}, year = {2011}, type = {Journal Article} } @article{Bauxbaum2015, author = {Buxbaum, A. R. and Haimovich, G. and Singer, R. H.}, title = {In the right place at the right time: visualizing and understanding mRNA localization}, journal = {Nature Reviews Molecular Cell Biology}, volume = {16}, number = {2}, pages = {95-109}, note = {Ca1ad Times Cited:5 Cited References Count:176}, abstract = {The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization.}, keywords = {yeast saccharomyces-cerevisiae green fluorescent protein in-vitro reconstitution zipcode-binding protein actin gene-expression xenopus-oocytes hippocampal-neurons endoplasmic-reticulum dendritic transport particle tracking}, ISSN = {1471-0072}, DOI = {10.1038/nrm3918}, url = {://WOS:000348643800010}, year = {2015}, type = {Journal Article} } @article{Dean2014, author = {Dean, K. M. and Palmer, A. E.}, title = {Advances in fluorescence labeling strategies for dynamic cellular imaging}, journal = {Nature Chemical Biology}, volume = {10}, number = {7}, pages = {512-523}, note = {Aj7iu Times Cited:22 Cited References Count:96}, abstract = {Synergistic advances in optical physics, probe design, molecular biology, labeling techniques and computational analysis have propelled fluorescence imaging into new realms of spatiotemporal resolution and sensitivity. This review aims to discuss advances in fluorescent probes and live-cell labeling strategies, two areas that remain pivotal for future advances in imaging technology. Fluorescent protein- and bio-orthogonal-based methods for protein and RNA imaging are discussed as well as emerging bioengineering techniques that enable their expression at specific genomic loci (for example, CRISPR and TALENs). Important attributes that contribute to the success of each technique are emphasized, providing a guideline for future advances in dynamic live-cell imaging.}, keywords = {directed tosyl chemistry zinc-finger nucleases living cells in-vivo superresolution microscopy bacterial phytochrome stokes shift live cells protein rna}, ISSN = {1552-4450}, DOI = {10.1038/Nchembio.1556}, url = {://WOS:000337871200008}, year = {2014}, type = {Journal Article} } @article{Fernandey-Suarez2008, author = {Fernandez-Suarez, M. and Ting, A. Y.}, title = {Fluorescent probes for super-resolution imaging in living cells}, journal = {Nat Rev Mol Cell Biol}, volume = {9}, number = {12}, pages = {929-43}, note = {Fernandez-Suarez, Marta Ting, Alice Y eng Review England 2008/11/13 09:00 Nat Rev Mol Cell Biol. 2008 Dec;9(12):929-43. doi: 10.1038/nrm2531. Epub 2008 Nov 12.}, abstract = {In 1873, Ernst Abbe discovered that features closer than approximately 200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1-5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.}, keywords = {Animals Carbocyanines/metabolism Cells/*metabolism Coloring Agents/metabolism Diagnostic Imaging/*methods Fluorescent Dyes/*metabolism Green Fluorescent Proteins/metabolism Luminescent Agents/metabolism Microscopy, Fluorescence/*methods}, ISSN = {1471-0080 (Electronic) 1471-0072 (Linking)}, DOI = {10.1038/nrm2531}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19002208}, year = {2008}, type = {Journal Article} } @book{Hartmann2009 author = {Hartmann, Roland K.}, title = {Handbook of RNA biochemistry}, publisher = {Wiley-VCH}, address = {Weinheim}, edition = {1st student}, note = {2009499142 edited by Roland K. Hartmann ... [et al.]. ill. (some col.) ; 25 cm. Includes bibliographical references and index.}, keywords = {rna.}, pages = {xliii, 931 p.}, ISBN = {9783527325344 (pbk. alk. paper)}, year = {2009}, type = {Book} } @article{Höfer2013, author = {Höfer, K. and Langejürgen, L. V. and Jäschke, A.}, title = {Universal aptamer-based real-time monitoring of enzymatic RNA synthesis}, journal = {J Am Chem Soc}, volume = {135}, number = {37}, pages = {13692-4}, note = {Hofer, Katharina Langejurgen, Lisa V Jaschke, Andres eng 2013/09/03 06:00 J Am Chem Soc. 2013 Sep 18;135(37):13692-4. doi: 10.1021/ja407142f. Epub 2013 Sep 4.}, abstract = {In vitro transcription is an essential laboratory technique for enzymatic RNA synthesis. Unfortunately, no methods exist for analyzing quality and quantity of the synthesized RNA while the transcription proceeds. Here we describe a simple, robust, and universal system for monitoring and quantifying the synthesis of any RNA in real time without interference from abortive transcription byproducts. The distinguishing feature is a universal fluorescence module (UFM), consisting of the eGFP-like Spinach aptamer and a highly active hammerhead ribozyme, which is appended to the RNA of interest (ROI). In the transcription mixture, the primary transcript is cleaved rapidly behind the ROI, thereby releasing always the same UFM, independent of the ROI sequence, polymerase, or promoter used. The UFM binds to the target of the Spinach aptamer, the fluorogenic dye DFHBI, and thereby induces a strong fluorescence signal. This design allows real-time quantification, standardization, parallelization, and high-throughput screening.}, keywords = {Aptamers, Nucleotide/*chemical synthesis/chemistry *Biological Assay Exodeoxyribonucleases/*metabolism Fluorescence}, ISSN = {1520-5126 (Electronic) 0002-7863 (Linking)}, DOI = {10.1021/ja407142f}, url = {http://www.ncbi.nlm.nih.gov/pubmed/23991672}, year = {2013}, type = {Journal Article} } @article{Kellenberger2015, author = {Kellenberger, C. A. and Chen, C. and Whiteley, A. T. and Portnoy, D. A. and Hammond, M. C.}, title = {RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP}, journal = {Journal of the American Chemical Society}, volume = {137}, number = {20}, pages = {6432-6435}, note = {Cj3kv Times Cited:0 Cited References Count:24}, abstract = {Cyclic di-AMP (cdiA) is a second messenger predicted, to be widespread in Gram-positive bacteria, some Gram-negative bacteria, and Archaea. In the human pathogen Listeria monocytogenes, cdiA is an essential molecule that regulates metabolic function and cell wall homeostasis, and decreased levels of cdiA result in increased antibiotic susceptibility. We have generated fluorescent biosensors for cdiA through fusion of the Spinach2 aptamer to ligand-binding domains of cdiA riboswitches. The biosensor was used to visualize intra-cellular cdiA levels in live L. monocytogenes strains and to determine the catalytic domain of the phosphodiesterase PdeA. Furthermore; a flow cytometry assay based On this biosensor was used to screen for diadenylate cyclase activity and confirmed the enzymatic activity of DisA-like proteins from Clostridium difficile and Methanocaldococcus jannaschii. Thus, We have expanded the development of RNA-based biosensors for in vivo metabolite imaging in Gram-positive bacteria and have validated the first dinucleotide cyclase from Archaea.}, keywords = {bacterial 2nd-messenger ydao riboswitch small molecules nucleotides virulence reveals enters sense gmp}, ISSN = {0002-7863}, DOI = {10.1021/jacs.5b00275}, url = {://WOS:000355383500003}, year = {2015}, type = {Journal Article} } @article{Kellenberber2013, author = {Kellenberger, C. A. and Wilson, S. C. and Sales-Lee, J. and Hammond, M. C.}, title = {RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messengers Cyclic di-GMP and Cyclic AMP-GMP}, journal = {Journal of the American Chemical Society}, volume = {135}, number = {13}, pages = {4906-4909}, note = {121QM Times Cited:32 Cited References Count:24}, abstract = {Cyclic dinucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed RNA-based fluorescent biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-1 riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic dinucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.}, keywords = {escherichia-coli ligand-binding riboswitch diguanylate magnesium bacteria cgmp DNA}, ISSN = {0002-7863}, DOI = {10.1021/ja311960g}, url = {://WOS:000317259300002}, year = {2013}, type = {Journal Article} } @article{Paige2011, author = {Paige, J. S. and Wu, K. Y. and Jaffrey, S. R.}, title = {RNA mimics of green fluorescent protein}, journal = {Science}, volume = {333}, number = {6042}, pages = {642-6}, note = {Paige, Jeremy S Wu, Karen Y Jaffrey, Samie R eng NS064516/NS/NINDS NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ R01 NS064516-03/NS/NINDS NIH HHS/ T32 CA062948/CA/NCI NIH HHS/ T32 CA062948-14/CA/NCI NIH HHS/ T32CA062948/CA/NCI NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't New York, N.Y. 2011/07/30 06:00 Science. 2011 Jul 29;333(6042):642-6. doi: 10.1126/science.1207339.}, abstract = {Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here, we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex, termed Spinach, resembles enhanced GFP and emits a green fluorescence comparable in brightness with fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.}, keywords = {Aptamers, Nucleotide/*chemistry/*metabolism Benzyl Compounds/*chemistry/*metabolism Biomimetics Cell Nucleus/metabolism Cytoplasmic Granules/metabolism Cytosol/metabolism *Fluorescence Green Fluorescent Proteins/*chemistry HEK293 Cells Humans Imidazolines/*chemistry/*metabolism Molecular Mimicry Nucleic Acid Conformation Photobleaching Protein Binding RNA, Untranslated/metabolism SELEX Aptamer Technique Spectrometry, Fluorescence Sucrose/pharmacology}, ISSN = {1095-9203 (Electronic) 0036-8075 (Linking)}, DOI = {10.1126/science.1207339}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21798953}, year = {2011}, type = {Journal Article} } @article{Pothoulakis2014, author = {Pothoulakis, G. and Ceroni, F. and Reeve, B. and Ellis, T.}, title = {The spinach RNA aptamer as a characterization tool for synthetic biology}, journal = {ACS Synth Biol}, volume = {3}, number = {3}, pages = {182-7}, note = {Pothoulakis, Georgios Ceroni, Francesca Reeve, Benjamin Ellis, Tom eng Research Support, Non-U.S. Gov't 2013/09/03 06:00 ACS Synth Biol. 2014 Mar 21;3(3):182-7. doi: 10.1021/sb400089c. Epub 2013 Sep 13.}, abstract = {Characterization of genetic control elements is essential for the predictable engineering of synthetic biology systems. The current standard for in vivo characterization of control elements is through the use of fluorescent reporter proteins such as green fluorescent protein (GFP). Gene expression, however, involves not only protein production but also the production of mRNA. Here, we present the use of the Spinach aptamer sequence, an RNA mimic of GFP, as a tool to characterize mRNA expression in Escherichia coli. We show how the aptamer can be incorporated into gene expression cassettes and how co-expressing it with a red fluorescent protein (mRFP1) allows, for the first time, simultaneous measurement of mRNA and protein levels from engineered constructs. Using flow cytometry, we apply this tool here to evaluate ribosome binding site sequences and promoters and use it to highlight the differences in the temporal behavior of transcription and translation.}, keywords = {Aptamers, Nucleotide/*genetics/metabolism Binding Sites Escherichia coli/genetics/metabolism Genetic Engineering/*methods Luminescent Proteins/genetics/metabolism RNA/*genetics/metabolism RNA, Bacterial/genetics/metabolism Spinacia oleracea/*genetics Synthetic Biology/*methods}, ISSN = {2161-5063 (Electronic) 2161-5063 (Linking)}, DOI = {10.1021/sb400089c}, url = {http://www.ncbi.nlm.nih.gov/pubmed/23991760}, year = {2014}, type = {Journal Article} } @article{Sassanfar1993, author = {Sassanfar, M. and Szostak, J. W.}, title = {An Rna Motif That Binds Atp}, journal = {Nature}, volume = {364}, number = {6437}, pages = {550-553}, note = {Lq667 Times Cited:389 Cited References Count:17}, abstract = {RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules1,2. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.}, keywords = {molecular recognition receptor}, ISSN = {0028-0836}, DOI = {Doi 10.1038/364550a0}, url = {://WOS:A1993LQ66700062}, year = {1993}, type = {Journal Article} } @article{Sastry1997, author = {Sastry, S. S. and Ross, B. M.}, title = {Nuclease activity of T7 RNA polymerase and the heterogeneity of transcription elongation complexes}, journal = {J Biol Chem}, volume = {272}, number = {13}, pages = {8644-52}, note = {Sastry, S S Ross, B M eng Research Support, Non-U.S. Gov't 1997/03/28 J Biol Chem. 1997 Mar 28;272(13):8644-52.}, abstract = {We have discovered that T7 RNA polymerase, purified to apparent homogeneity from overexpressing Escherichia coli cells, possesses a DNase and an RNase activity. Mutations in the active center of T7 RNA polymerase abolished or greatly decreased the nuclease activity. This nuclease activity is specific for single-stranded DNA and RNA oligonucleotides and does not manifest on double-stranded DNAs. Under the conditions of promoter-driven transcription on double-stranded DNA, no nuclease activity was observed. The nuclease attacks DNA oligonucleotides in mono- or dinucleotide steps. The nuclease is a 3' to 5' exonuclease leaving a 3'-OH end, and it degrades DNA oligonucleotides to a minimum size of 3 to 5 nucleotides. It is completely dependent on Mg2+. The T7 RNA polymerase-nuclease is inhibited by T7 lysozyme and heparin, although not completely. In the presence of rNTPs, the nuclease activity is suppressed but an unusual 3'-end-initiated polymerase activity is unmasked. RNA from isolated pre-elongation and elongation complexes arrested by a psoralen roadblock or naturally paused at the 3'-end of an oligonucleotide template exhibited evidence of nuclease activity. The nuclease activity of T7 RNA polymerase is unrelated to pyrophosphorolysis. We propose that the nuclease of T7 RNA polymerase acts only in arrested or paused elongation complexes, and that in combination with the unusual 3'-end polymerizing activity, causes heterogeneity in elongation complexes. Additionally, during normal transcription elongation, the kinetic balance between nuclease and polymerase is shifted in favor of polymerase.}, keywords = {*Bacteriophage T7 Base Sequence DNA, Single-Stranded/metabolism DNA-Directed RNA Polymerases/*metabolism Deoxyribonucleases/metabolism Electrophoresis, Polyacrylamide Gel Enzyme Activation Furocoumarins/metabolism Heparin/pharmacology Magnesium/metabolism Molecular Sequence Data N-Acetylmuramoyl-L-alanine Amidase/metabolism *Peptide Elongation Factors RNA/metabolism Ribonucleases/metabolism Transcription, Genetic Viral Proteins}, ISSN = {0021-9258 (Print) 0021-9258 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/9079696}, year = {1997}, type = {Journal Article} } @article{Strack2013, author = {Strack, R. L. and Disney, M. D. and Jaffrey, S. R.}, title = {A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA}, journal = {Nat Methods}, volume = {10}, number = {12}, pages = {1219-24}, note = {Strack, Rita L Disney, Matthew D Jaffrey, Samie R eng F32 GM106683/GM/NIGMS NIH HHS/ GM079235/GM/NIGMS NIH HHS/ NS010249/NS/NINDS NIH HHS/ R01 EB010249/EB/NIBIB NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ Research Support, N.I.H., Extramural 2013/10/29 06:00 Nat Methods. 2013 Dec;10(12):1219-24. doi: 10.1038/nmeth.2701. Epub 2013 Oct 27.}, abstract = {Imaging RNA in living cells is a challenging problem in cell biology. One strategy for genetically encoding fluorescent RNAs is to express them as fusions with Spinach, an 'RNA mimic of GFP'. We found that Spinach was dimmer than expected when used to tag constructs in living cells owing to a combination of thermal instability and a propensity for misfolding. Using systematic mutagenesis, we generated Spinach2 that overcomes these issues and can be used to image diverse RNAs. Using Spinach2, we detailed the dynamics of the CGG trinucleotide repeat-containing 'toxic RNA' associated with Fragile X-associated tremor/ataxia syndrome, and show that these RNAs form nuclear foci with unexpected morphological plasticity that is regulated by the cell cycle and by small molecules. Together, these data demonstrate that Spinach2 exhibits improved versatility for fluorescently labeling RNAs in living cells.}, keywords = {Animals Base Sequence COS Cells Cercopithecus aethiops DNA Mutational Analysis Escherichia coli/metabolism Fluorescent Dyes/chemistry Green Fluorescent Proteins/metabolism HEK293 Cells Humans Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Protein Denaturation Protein Folding RNA/*chemistry RNA, Untranslated/*chemistry Temperature *Trinucleotide Repeats}, ISSN = {1548-7105 (Electronic) 1548-7091 (Linking)}, DOI = {10.1038/nmeth.2701}, url = {http://www.ncbi.nlm.nih.gov/pubmed/24162923}, year = {2013}, type = {Journal Article} } @article{Strack2015, author = {Strack, R. L. and Jaffrey, S. R.}, title = {Live-cell imaging of mammalian RNAs with Spinach2}, journal = {Methods Enzymol}, volume = {550}, pages = {129-46}, note = {Strack, Rita L Jaffrey, Samie R eng F32 GM106683/GM/NIGMS NIH HHS/ NS010249/NS/NINDS NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ Research Support, N.I.H., Extramural 2015/01/22 06:00 Methods Enzymol. 2015;550:129-46. doi: 10.1016/bs.mie.2014.10.044. Epub 2015 Jan 6.}, abstract = {The ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of imaging individual RNAs of interest in living cells, each with their own advantages and limitations. This chapter provides an overview of current methods of live-cell RNA imaging, including a detailed discussion of genetically encoded strategies for labeling RNAs in mammalian cells. This chapter then focuses on the development and use of "RNA mimics of GFP" or Spinach technology for tagging mammalian RNAs and includes a detailed protocol for imaging 5S and CGG60 RNA with the recently described Spinach2 tag.}, ISSN = {1557-7988 (Electronic) 0076-6879 (Linking)}, DOI = {10.1016/bs.mie.2014.10.044}, url = {http://www.ncbi.nlm.nih.gov/pubmed/25605384}, year = {2015}, type = {Journal Article} } @article{Tyagi2009, author = {Tyagi, S.}, title = {Imaging intracellular RNA distribution and dynamics in living cells}, journal = {Nat Methods}, volume = {6}, number = {5}, pages = {331-8}, note = {Tyagi, Sanjay eng MH079197/MH/NIMH NIH HHS/ Research Support, N.I.H., Extramural Review 2009/05/01 09:00 Nat Methods. 2009 May;6(5):331-8. doi: 10.1038/nmeth.1321.}, abstract = {Powerful methods now allow the imaging of specific mRNAs in living cells. These methods enlist fluorescent proteins to illuminate mRNAs, use labeled oligonucleotide probes and exploit aptamers that render organic dyes fluorescent. The intracellular dynamics of mRNA synthesis, transport and localization can be analyzed at higher temporal resolution with these methods than has been possible with traditional fixed-cell or biochemical approaches. These methods have also been adopted to visualize and track single mRNA molecules in real time. This review explores the promises and limitations of these methods.}, keywords = {Aptamers, Nucleotide/chemistry Cell Survival Cells/*cytology/*metabolism Cytophotometry/*methods Fluorescent Dyes/chemistry Green Fluorescent Proteins/chemistry/genetics Microscopy, Fluorescence/methods Oligonucleotide Probes/chemistry RNA, Messenger/*analysis/chemistry/*metabolism RNA-Binding Proteins/chemistry/genetics Recombinant Fusion Proteins/chemistry/genetics}, ISSN = {1548-7105 (Electronic) 1548-7091 (Linking)}, DOI = {10.1038/nmeth.1321}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19404252}, year = {2009}, type = {Journal Article} } year = {2015} } @article{Li2013, abstract = {We designed a novel aptamer based biosensor (aptasensor) for ultrasensitive detection of adenosine triphosphate (ATP) through resonance energy transfer (RET). The ATP aptamer was modified with Cy3 at the 3' end, and a green quantum dot (525) was attached to the 5' end of its complementary sequence respectively. The ATP aptamer and its complementary sequence could assemble into a duplex structure in the absence of target ATP, and then decrease the distance between the quantum dot and Cy3 which could produce significant RET signal. Upon ATP binding, the ATP aptamer could dissociate with its complementary sequence and then increase the distance between the quantum dot and Cy3 which would significantly decrease the RET signal. Therefore, the ATP detection could be easily achieved through detection of the fluorescence intensity ratio between 525 nm and 560 nm. The results show that the emission fluorescence intensity ratio of 525/560 is linearly related to the logarithmic concentration of ATP. The linear range of this aptasensor is from 0.1 nM to 1 $\mu$M, and the detection limit is lower down to 0.01 nM. Excellent selectivity of this aptasensor for ATP has been demonstrated through the detection of thymidine triphosphate (TTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP) and adenosine diphosphate (ADP) respectively as control. The method we described here could easily detect ATP with excellent selectivity, linearity and sensitivity down to the nanomolar range, as well as avoid photobleaching.}, author = {Li, Zheng and Wang, Yijing and Liu, Ying and Zeng, Yongyi and Huang, Aimin and Peng, Niancai and Liu, Xiaolong and Liu, Jingfeng}, doi = {10.1039/c3an00449j}, file = {:root/Downloads/c3an00449j.pdf:pdf}, isbn = {1364-5528 (Electronic)$\backslash$r0003-2654 (Linking)}, issn = {1364-5528}, journal = {The Analyst}, keywords = {Adenosine Triphosphate,Adenosine Triphosphate: analysis,Adenosine Triphosphate: metabolism,Aptamers, Nucleotide,Aptamers, Nucleotide: genetics,Aptamers, Nucleotide: metabolism,Base Sequence,Biosensing Techniques,Biosensing Techniques: methods,Fluorescence Resonance Energy Transfer,Quantum Dots}, number = {17}, pages = {4732--6}, pmid = {23814782}, title = {{A novel aptasensor for the ultra-sensitive detection of adenosine triphosphate via aptamer/quantum dot based resonance energy transfer.}}, url = {http://www.ncbi.nlm.nih.gov/pubmed/23814782}, volume = {138}, year = {2013} } @article{Rivas2015, author = {Rivas, Lourdes and Mayorga-Martinez, Carmen C. and Quesada-Gonz\'{a}lez, Daniel and Zamora-G\'{a}lvez, Alejandro and de la Escosura-Mu\~{n}iz, Alfredo and Merko\c{c}i, Arben}, doi = {10.1021/acs.analchem.5b00890}, file = {:root/Downloads/acs\%2Eanalchem\%2E5b00890.pdf:pdf}, issn = {0003-2700}, journal = {Analytical Chemistry}, pages = {150501134921005}, title = {{Label-Free Impedimetric Aptasensor for Ochratoxin-A Detection Using Iridium Oxide Nanoparticles}}, url = {http://pubs.acs.org/doi/abs/10.1021/acs.analchem.5b00890}, year = {2015} }@article{Balbo2007, author = {Balbo, P. B. and Bohm, A.}, title = {Mechanism of poly(A) polymerase: structure of the enzyme-MgATP-RNA ternary complex and kinetic analysis}, journal = {Structure}, volume = {15}, number = {9}, pages = {1117-31}, note = {Balbo, Paul B Bohm, Andrew eng GM 065972/GM/NIGMS NIH HHS/ R01 GM065972/GM/NIGMS NIH HHS/ R01 GM065972-05/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural London, England : 1993 2007/09/14 09:00 Structure. 2007 Sep;15(9):1117-31.}, abstract = {We report the 1.8 A structure of yeast poly(A) polymerase (PAP) trapped in complex with ATP and a five residue poly(A) by mutation of the catalytically required aspartic acid 154 to alanine. The enzyme has undergone significant domain movement and reveals a closed conformation with extensive interactions between the substrates and all three polymerase domains. Both substrates and 31 buried water molecules are enclosed within a central cavity that is open at both ends. Four PAP mutants were subjected to detailed kinetic analysis, and studies of the adenylyltransfer (forward), pyrophosphorolysis (reverse), and nucleotidyltransfer reaction utilizing CTP for the mutants are presented. The results support a model in which binding of both poly(A) and the correct nucleotide, MgATP, induces a conformational change, resulting in formation of a stable, closed enzyme state. Thermodynamic considerations of the data are discussed as they pertain to domain closure, substrate specificity, and catalytic strategies utilized by PAP.}, keywords = {Adenosine Triphosphate/*metabolism Catalysis Kinetics Models, Molecular Mutagenesis, Site-Directed Polynucleotide Adenylyltransferase/chemistry/genetics/*metabolism Protein Conformation RNA/chemistry/*metabolism}, ISSN = {0969-2126 (Print) 0969-2126 (Linking)}, DOI = {10.1016/j.str.2007.07.010}, url = {http://www.ncbi.nlm.nih.gov/pubmed/17850751}, year = {2007}, type = {Journal Article} } @article{Baugh2000, author = {Baugh, C. and Grate, D. and Wilson, C.}, title = {2.8 angstrom crystal structure of the malachite green aptamer}, journal = {Journal of Molecular Biology}, volume = {301}, number = {1}, pages = {117-128}, note = {343MC Times Cited:90 Cited References Count:52}, abstract = {Previous in vitro selection experiments identified an RNA aptamer that recognizes the chromophore malachite green (MG) with a high level of affinity, and which undergoes site-specific cleavage following laser irradiation. To understand the mechanism by which this RNA folds to recognize specifically its ligand and the structural basis for chromophore-assisted laser inactivation, we have determined the 2.8 Angstrom crystal structure of the aptamer bound to tetramethylrosamine (TMR), a high-affinity MG analog. The ligand-binding site is defined by an asymmetric internal loop, flanked by a pair of helices. A U-turn and several non-canonical base interactions stabilize the folding of loop nucleotides around the TMR. The aptamer utilizes several tiers of stacked nucleotides arranged in pairs, triples, and a novel base quadruple to effectively encapsulate the ligand. Even in the absence of specific stabilizing hydrogen bonds, discrimination between related fluorophores and chromophores is possible due to tight packing in the RNA binding pocket, which severely limits the size and shape of recognized ligands. The site of laser-induced cleavage lies relatively far from the bound TMR (similar to 15 Angstrom). The unusual backbone conformation of the cleavage site nucleotide and its high level of solvent accessibility may, combine to allow preferential reaction with freely diffusing hydroxyl radicals generated at the bound ligand. Several observations, however, favor alternative mechanisms for cleavage, such as conformational changes in the aptamer or long-range electron transfer between the bound ligand and the cleavage site nucleotide. (C) 2000 Academic Press.}, keywords = {in vitro selection tetramethylrosamine base quadruple chromophore-assisted laser inactivation assisted laser inactivation ribosomal-rna binding rna DNA recognition resolution complex discrimination selection molecules}, ISSN = {0022-2836}, DOI = {10.1006/jmbi.2000.3951}, url = {://WOS:000088705300010}, year = {2000}, type = {Journal Article} } @article{Beckert2011, author = {Beckert, B. and Masquida, B.}, title = {Synthesis of RNA by in vitro transcription}, journal = {Methods Mol Biol}, volume = {703}, pages = {29-41}, note = {Beckert, Bertrand Masquida, Benoit eng Clifton, N.J. 2010/12/03 06:00 Methods Mol Biol. 2011;703:29-41. doi: 10.1007/978-1-59745-248-9_3.}, abstract = {In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases in mug to mg quantities. It is based on the engineering of a template that includes a bacteriophage promoter sequence (e.g. from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase. In vitro transcripts are used in analytical techniques (e.g. hybridization analysis), structural studies (for NMR and X-ray crystallography), in biochemical and genetic studies (e.g. as antisense reagents), and as functional molecules (ribozymes and aptamers).}, keywords = {Bacteriophage T7/genetics Base Sequence DNA-Directed RNA Polymerases/genetics In Vitro Techniques Molecular Biology/*methods Molecular Sequence Data Promoter Regions, Genetic/genetics RNA/*chemical synthesis *Transcription, Genetic}, ISSN = {1940-6029 (Electronic) 1064-3745 (Linking)}, DOI = {10.1007/978-1-59745-248-9_3}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21125481}, year = {2011}, type = {Journal Article} } @article{Bauxbaum2015, author = {Buxbaum, A. R. and Haimovich, G. and Singer, R. H.}, title = {In the right place at the right time: visualizing and understanding mRNA localization}, journal = {Nature Reviews Molecular Cell Biology}, volume = {16}, number = {2}, pages = {95-109}, note = {Ca1ad Times Cited:5 Cited References Count:176}, abstract = {The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization.}, keywords = {yeast saccharomyces-cerevisiae green fluorescent protein in-vitro reconstitution zipcode-binding protein actin gene-expression xenopus-oocytes hippocampal-neurons endoplasmic-reticulum dendritic transport particle tracking}, ISSN = {1471-0072}, DOI = {10.1038/nrm3918}, url = {://WOS:000348643800010}, year = {2015}, type = {Journal Article} } @article{Dean2014, author = {Dean, K. M. and Palmer, A. E.}, title = {Advances in fluorescence labeling strategies for dynamic cellular imaging}, journal = {Nature Chemical Biology}, volume = {10}, number = {7}, pages = {512-523}, note = {Aj7iu Times Cited:22 Cited References Count:96}, abstract = {Synergistic advances in optical physics, probe design, molecular biology, labeling techniques and computational analysis have propelled fluorescence imaging into new realms of spatiotemporal resolution and sensitivity. This review aims to discuss advances in fluorescent probes and live-cell labeling strategies, two areas that remain pivotal for future advances in imaging technology. Fluorescent protein- and bio-orthogonal-based methods for protein and RNA imaging are discussed as well as emerging bioengineering techniques that enable their expression at specific genomic loci (for example, CRISPR and TALENs). Important attributes that contribute to the success of each technique are emphasized, providing a guideline for future advances in dynamic live-cell imaging.}, keywords = {directed tosyl chemistry zinc-finger nucleases living cells in-vivo superresolution microscopy bacterial phytochrome stokes shift live cells protein rna}, ISSN = {1552-4450}, DOI = {10.1038/Nchembio.1556}, url = {://WOS:000337871200008}, year = {2014}, type = {Journal Article} } @article{Fernandey-Suarez2008, author = {Fernandez-Suarez, M. and Ting, A. Y.}, title = {Fluorescent probes for super-resolution imaging in living cells}, journal = {Nat Rev Mol Cell Biol}, volume = {9}, number = {12}, pages = {929-43}, note = {Fernandez-Suarez, Marta Ting, Alice Y eng Review England 2008/11/13 09:00 Nat Rev Mol Cell Biol. 2008 Dec;9(12):929-43. doi: 10.1038/nrm2531. Epub 2008 Nov 12.}, abstract = {In 1873, Ernst Abbe discovered that features closer than approximately 200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1-5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.}, keywords = {Animals Carbocyanines/metabolism Cells/*metabolism Coloring Agents/metabolism Diagnostic Imaging/*methods Fluorescent Dyes/*metabolism Green Fluorescent Proteins/metabolism Luminescent Agents/metabolism Microscopy, Fluorescence/*methods}, ISSN = {1471-0080 (Electronic) 1471-0072 (Linking)}, DOI = {10.1038/nrm2531}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19002208}, year = {2008}, type = {Journal Article} } @book{Hartmann2009 author = {Hartmann, Roland K.}, title = {Handbook of RNA biochemistry}, publisher = {Wiley-VCH}, address = {Weinheim}, edition = {1st student}, note = {2009499142 edited by Roland K. Hartmann ... [et al.]. ill. (some col.) ; 25 cm. Includes bibliographical references and index.}, keywords = {rna.}, pages = {xliii, 931 p.}, ISBN = {9783527325344 (pbk. alk. paper)}, year = {2009}, type = {Book} } @article{Höfer2013, author = {Höfer, K. and Langejürgen, L. V. and Jäschke, A.}, title = {Universal aptamer-based real-time monitoring of enzymatic RNA synthesis}, journal = {J Am Chem Soc}, volume = {135}, number = {37}, pages = {13692-4}, note = {Hofer, Katharina Langejurgen, Lisa V Jaschke, Andres eng 2013/09/03 06:00 J Am Chem Soc. 2013 Sep 18;135(37):13692-4. doi: 10.1021/ja407142f. Epub 2013 Sep 4.}, abstract = {In vitro transcription is an essential laboratory technique for enzymatic RNA synthesis. Unfortunately, no methods exist for analyzing quality and quantity of the synthesized RNA while the transcription proceeds. Here we describe a simple, robust, and universal system for monitoring and quantifying the synthesis of any RNA in real time without interference from abortive transcription byproducts. The distinguishing feature is a universal fluorescence module (UFM), consisting of the eGFP-like Spinach aptamer and a highly active hammerhead ribozyme, which is appended to the RNA of interest (ROI). In the transcription mixture, the primary transcript is cleaved rapidly behind the ROI, thereby releasing always the same UFM, independent of the ROI sequence, polymerase, or promoter used. The UFM binds to the target of the Spinach aptamer, the fluorogenic dye DFHBI, and thereby induces a strong fluorescence signal. This design allows real-time quantification, standardization, parallelization, and high-throughput screening.}, keywords = {Aptamers, Nucleotide/*chemical synthesis/chemistry *Biological Assay Exodeoxyribonucleases/*metabolism Fluorescence}, ISSN = {1520-5126 (Electronic) 0002-7863 (Linking)}, DOI = {10.1021/ja407142f}, url = {http://www.ncbi.nlm.nih.gov/pubmed/23991672}, year = {2013}, type = {Journal Article} } @article{Kellenberger2015, author = {Kellenberger, C. A. and Chen, C. and Whiteley, A. T. and Portnoy, D. A. and Hammond, M. C.}, title = {RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP}, journal = {Journal of the American Chemical Society}, volume = {137}, number = {20}, pages = {6432-6435}, note = {Cj3kv Times Cited:0 Cited References Count:24}, abstract = {Cyclic di-AMP (cdiA) is a second messenger predicted, to be widespread in Gram-positive bacteria, some Gram-negative bacteria, and Archaea. In the human pathogen Listeria monocytogenes, cdiA is an essential molecule that regulates metabolic function and cell wall homeostasis, and decreased levels of cdiA result in increased antibiotic susceptibility. We have generated fluorescent biosensors for cdiA through fusion of the Spinach2 aptamer to ligand-binding domains of cdiA riboswitches. The biosensor was used to visualize intra-cellular cdiA levels in live L. monocytogenes strains and to determine the catalytic domain of the phosphodiesterase PdeA. Furthermore; a flow cytometry assay based On this biosensor was used to screen for diadenylate cyclase activity and confirmed the enzymatic activity of DisA-like proteins from Clostridium difficile and Methanocaldococcus jannaschii. Thus, We have expanded the development of RNA-based biosensors for in vivo metabolite imaging in Gram-positive bacteria and have validated the first dinucleotide cyclase from Archaea.}, keywords = {bacterial 2nd-messenger ydao riboswitch small molecules nucleotides virulence reveals enters sense gmp}, ISSN = {0002-7863}, DOI = {10.1021/jacs.5b00275}, url = {://WOS:000355383500003}, year = {2015}, type = {Journal Article} } @article{Kellenberber2013, author = {Kellenberger, C. A. and Wilson, S. C. and Sales-Lee, J. and Hammond, M. C.}, title = {RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messengers Cyclic di-GMP and Cyclic AMP-GMP}, journal = {Journal of the American Chemical Society}, volume = {135}, number = {13}, pages = {4906-4909}, note = {121QM Times Cited:32 Cited References Count:24}, abstract = {Cyclic dinucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed RNA-based fluorescent biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-1 riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic dinucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.}, keywords = {escherichia-coli ligand-binding riboswitch diguanylate magnesium bacteria cgmp DNA}, ISSN = {0002-7863}, DOI = {10.1021/ja311960g}, url = {://WOS:000317259300002}, year = {2013}, type = {Journal Article} } @article{Paige2011, author = {Paige, J. S. and Wu, K. Y. and Jaffrey, S. R.}, title = {RNA mimics of green fluorescent protein}, journal = {Science}, volume = {333}, number = {6042}, pages = {642-6}, note = {Paige, Jeremy S Wu, Karen Y Jaffrey, Samie R eng NS064516/NS/NINDS NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ R01 NS064516-03/NS/NINDS NIH HHS/ T32 CA062948/CA/NCI NIH HHS/ T32 CA062948-14/CA/NCI NIH HHS/ T32CA062948/CA/NCI NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't New York, N.Y. 2011/07/30 06:00 Science. 2011 Jul 29;333(6042):642-6. doi: 10.1126/science.1207339.}, abstract = {Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here, we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex, termed Spinach, resembles enhanced GFP and emits a green fluorescence comparable in brightness with fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.}, keywords = {Aptamers, Nucleotide/*chemistry/*metabolism Benzyl Compounds/*chemistry/*metabolism Biomimetics Cell Nucleus/metabolism Cytoplasmic Granules/metabolism Cytosol/metabolism *Fluorescence Green Fluorescent Proteins/*chemistry HEK293 Cells Humans Imidazolines/*chemistry/*metabolism Molecular Mimicry Nucleic Acid Conformation Photobleaching Protein Binding RNA, Untranslated/metabolism SELEX Aptamer Technique Spectrometry, Fluorescence Sucrose/pharmacology}, ISSN = {1095-9203 (Electronic) 0036-8075 (Linking)}, DOI = {10.1126/science.1207339}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21798953}, year = {2011}, type = {Journal Article} } @article{Pothoulakis2014, author = {Pothoulakis, G. and Ceroni, F. and Reeve, B. and Ellis, T.}, title = {The spinach RNA aptamer as a characterization tool for synthetic biology}, journal = {ACS Synth Biol}, volume = {3}, number = {3}, pages = {182-7}, note = {Pothoulakis, Georgios Ceroni, Francesca Reeve, Benjamin Ellis, Tom eng Research Support, Non-U.S. Gov't 2013/09/03 06:00 ACS Synth Biol. 2014 Mar 21;3(3):182-7. doi: 10.1021/sb400089c. Epub 2013 Sep 13.}, abstract = {Characterization of genetic control elements is essential for the predictable engineering of synthetic biology systems. The current standard for in vivo characterization of control elements is through the use of fluorescent reporter proteins such as green fluorescent protein (GFP). Gene expression, however, involves not only protein production but also the production of mRNA. Here, we present the use of the Spinach aptamer sequence, an RNA mimic of GFP, as a tool to characterize mRNA expression in Escherichia coli. We show how the aptamer can be incorporated into gene expression cassettes and how co-expressing it with a red fluorescent protein (mRFP1) allows, for the first time, simultaneous measurement of mRNA and protein levels from engineered constructs. Using flow cytometry, we apply this tool here to evaluate ribosome binding site sequences and promoters and use it to highlight the differences in the temporal behavior of transcription and translation.}, keywords = {Aptamers, Nucleotide/*genetics/metabolism Binding Sites Escherichia coli/genetics/metabolism Genetic Engineering/*methods Luminescent Proteins/genetics/metabolism RNA/*genetics/metabolism RNA, Bacterial/genetics/metabolism Spinacia oleracea/*genetics Synthetic Biology/*methods}, ISSN = {2161-5063 (Electronic) 2161-5063 (Linking)}, DOI = {10.1021/sb400089c}, url = {http://www.ncbi.nlm.nih.gov/pubmed/23991760}, year = {2014}, type = {Journal Article} } @article{Sassanfar1993, author = {Sassanfar, M. and Szostak, J. W.}, title = {An Rna Motif That Binds Atp}, journal = {Nature}, volume = {364}, number = {6437}, pages = {550-553}, note = {Lq667 Times Cited:389 Cited References Count:17}, abstract = {RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules1,2. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.}, keywords = {molecular recognition receptor}, ISSN = {0028-0836}, DOI = {Doi 10.1038/364550a0}, url = {://WOS:A1993LQ66700062}, year = {1993}, type = {Journal Article} } @article{Sastry1997, author = {Sastry, S. S. and Ross, B. M.}, title = {Nuclease activity of T7 RNA polymerase and the heterogeneity of transcription elongation complexes}, journal = {J Biol Chem}, volume = {272}, number = {13}, pages = {8644-52}, note = {Sastry, S S Ross, B M eng Research Support, Non-U.S. Gov't 1997/03/28 J Biol Chem. 1997 Mar 28;272(13):8644-52.}, abstract = {We have discovered that T7 RNA polymerase, purified to apparent homogeneity from overexpressing Escherichia coli cells, possesses a DNase and an RNase activity. Mutations in the active center of T7 RNA polymerase abolished or greatly decreased the nuclease activity. This nuclease activity is specific for single-stranded DNA and RNA oligonucleotides and does not manifest on double-stranded DNAs. Under the conditions of promoter-driven transcription on double-stranded DNA, no nuclease activity was observed. The nuclease attacks DNA oligonucleotides in mono- or dinucleotide steps. The nuclease is a 3' to 5' exonuclease leaving a 3'-OH end, and it degrades DNA oligonucleotides to a minimum size of 3 to 5 nucleotides. It is completely dependent on Mg2+. The T7 RNA polymerase-nuclease is inhibited by T7 lysozyme and heparin, although not completely. In the presence of rNTPs, the nuclease activity is suppressed but an unusual 3'-end-initiated polymerase activity is unmasked. RNA from isolated pre-elongation and elongation complexes arrested by a psoralen roadblock or naturally paused at the 3'-end of an oligonucleotide template exhibited evidence of nuclease activity. The nuclease activity of T7 RNA polymerase is unrelated to pyrophosphorolysis. We propose that the nuclease of T7 RNA polymerase acts only in arrested or paused elongation complexes, and that in combination with the unusual 3'-end polymerizing activity, causes heterogeneity in elongation complexes. Additionally, during normal transcription elongation, the kinetic balance between nuclease and polymerase is shifted in favor of polymerase.}, keywords = {*Bacteriophage T7 Base Sequence DNA, Single-Stranded/metabolism DNA-Directed RNA Polymerases/*metabolism Deoxyribonucleases/metabolism Electrophoresis, Polyacrylamide Gel Enzyme Activation Furocoumarins/metabolism Heparin/pharmacology Magnesium/metabolism Molecular Sequence Data N-Acetylmuramoyl-L-alanine Amidase/metabolism *Peptide Elongation Factors RNA/metabolism Ribonucleases/metabolism Transcription, Genetic Viral Proteins}, ISSN = {0021-9258 (Print) 0021-9258 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/9079696}, year = {1997}, type = {Journal Article} } @article{Strack2013, author = {Strack, R. L. and Disney, M. D. and Jaffrey, S. R.}, title = {A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA}, journal = {Nat Methods}, volume = {10}, number = {12}, pages = {1219-24}, note = {Strack, Rita L Disney, Matthew D Jaffrey, Samie R eng F32 GM106683/GM/NIGMS NIH HHS/ GM079235/GM/NIGMS NIH HHS/ NS010249/NS/NINDS NIH HHS/ R01 EB010249/EB/NIBIB NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ Research Support, N.I.H., Extramural 2013/10/29 06:00 Nat Methods. 2013 Dec;10(12):1219-24. doi: 10.1038/nmeth.2701. Epub 2013 Oct 27.}, abstract = {Imaging RNA in living cells is a challenging problem in cell biology. One strategy for genetically encoding fluorescent RNAs is to express them as fusions with Spinach, an 'RNA mimic of GFP'. We found that Spinach was dimmer than expected when used to tag constructs in living cells owing to a combination of thermal instability and a propensity for misfolding. Using systematic mutagenesis, we generated Spinach2 that overcomes these issues and can be used to image diverse RNAs. Using Spinach2, we detailed the dynamics of the CGG trinucleotide repeat-containing 'toxic RNA' associated with Fragile X-associated tremor/ataxia syndrome, and show that these RNAs form nuclear foci with unexpected morphological plasticity that is regulated by the cell cycle and by small molecules. Together, these data demonstrate that Spinach2 exhibits improved versatility for fluorescently labeling RNAs in living cells.}, keywords = {Animals Base Sequence COS Cells Cercopithecus aethiops DNA Mutational Analysis Escherichia coli/metabolism Fluorescent Dyes/chemistry Green Fluorescent Proteins/metabolism HEK293 Cells Humans Molecular Sequence Data Mutagenesis Nucleic Acid Conformation Protein Denaturation Protein Folding RNA/*chemistry RNA, Untranslated/*chemistry Temperature *Trinucleotide Repeats}, ISSN = {1548-7105 (Electronic) 1548-7091 (Linking)}, DOI = {10.1038/nmeth.2701}, url = {http://www.ncbi.nlm.nih.gov/pubmed/24162923}, year = {2013}, type = {Journal Article} } @article{Strack2015, author = {Strack, R. L. and Jaffrey, S. R.}, title = {Live-cell imaging of mammalian RNAs with Spinach2}, journal = {Methods Enzymol}, volume = {550}, pages = {129-46}, note = {Strack, Rita L Jaffrey, Samie R eng F32 GM106683/GM/NIGMS NIH HHS/ NS010249/NS/NINDS NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ Research Support, N.I.H., Extramural 2015/01/22 06:00 Methods Enzymol. 2015;550:129-46. doi: 10.1016/bs.mie.2014.10.044. Epub 2015 Jan 6.}, abstract = {The ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of imaging individual RNAs of interest in living cells, each with their own advantages and limitations. This chapter provides an overview of current methods of live-cell RNA imaging, including a detailed discussion of genetically encoded strategies for labeling RNAs in mammalian cells. This chapter then focuses on the development and use of "RNA mimics of GFP" or Spinach technology for tagging mammalian RNAs and includes a detailed protocol for imaging 5S and CGG60 RNA with the recently described Spinach2 tag.}, ISSN = {1557-7988 (Electronic) 0076-6879 (Linking)}, DOI = {10.1016/bs.mie.2014.10.044}, url = {http://www.ncbi.nlm.nih.gov/pubmed/25605384}, year = {2015}, type = {Journal Article} } @article{Tyagi2009, author = {Tyagi, S.}, title = {Imaging intracellular RNA distribution and dynamics in living cells}, journal = {Nat Methods}, volume = {6}, number = {5}, pages = {331-8}, note = {Tyagi, Sanjay eng MH079197/MH/NIMH NIH HHS/ Research Support, N.I.H., Extramural Review 2009/05/01 09:00 Nat Methods. 2009 May;6(5):331-8. doi: 10.1038/nmeth.1321.}, abstract = {Powerful methods now allow the imaging of specific mRNAs in living cells. These methods enlist fluorescent proteins to illuminate mRNAs, use labeled oligonucleotide probes and exploit aptamers that render organic dyes fluorescent. The intracellular dynamics of mRNA synthesis, transport and localization can be analyzed at higher temporal resolution with these methods than has been possible with traditional fixed-cell or biochemical approaches. These methods have also been adopted to visualize and track single mRNA molecules in real time. This review explores the promises and limitations of these methods.}, keywords = {Aptamers, Nucleotide/chemistry Cell Survival Cells/*cytology/*metabolism Cytophotometry/*methods Fluorescent Dyes/chemistry Green Fluorescent Proteins/chemistry/genetics Microscopy, Fluorescence/methods Oligonucleotide Probes/chemistry RNA, Messenger/*analysis/chemistry/*metabolism RNA-Binding Proteins/chemistry/genetics Recombinant Fusion Proteins/chemistry/genetics}, ISSN = {1548-7105 (Electronic) 1548-7091 (Linking)}, DOI = {10.1038/nmeth.1321}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19404252}, year = {2009}, type = {Journal Article} } @Electronic{igemHD2015, title = {iGEM Heidelberg 2015 Official Wiki}, author = {iGEM Team Heidelberg}, howpublished = {http://2015.igem.org/Team:Heidelberg}, } @article{Bagby2009, author = {Bagby, S. C. and Bergman, N. H. and Shechner, D. M. and Yen, C. and Bartel, D. P.}, title = {A class I ligase ribozyme with reduced Mg2+ dependence: Selection, sequence analysis, and identification of functional tertiary interactions}, journal = {RNA}, volume = {15}, number = {12}, pages = {2129-46}, note = {Bagby, Sarah C Bergman, Nicholas H Shechner, David M Yen, Catherine Bartel, David P eng GM61835/GM/NIGMS NIH HHS/ R01 GM061835/GM/NIGMS NIH HHS/ Howard Hughes Medical Institute/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't New York, N.Y. 2009/12/01 06:00 RNA. 2009 Dec;15(12):2129-46. doi: 10.1261/rna.1912509.}, abstract = {The class I ligase was among the first ribozymes to have been isolated from random sequences and represents the catalytic core of several RNA-directed RNA polymerase ribozymes. The ligase is also notable for its catalytic efficiency and structural complexity. Here, we report an improved version of this ribozyme, arising from selection that targeted the kinetics of the chemical step. Compared with the parent ribozyme, the improved ligase achieves a modest increase in rate enhancement under the selective conditions and shows a sharp reduction in [Mg(2+)] dependence. Analysis of the sequences and kinetics of successful clones suggests which mutations play the greatest part in these improvements. Moreover, backbone and nucleobase interference maps of the parent and improved ligase ribozymes complement the newly solved crystal structure of the improved ligase to identify the functionally significant interactions underlying the catalytic ability and structural complexity of the ligase ribozyme.}, keywords = {Base Sequence Binding Sites Crystallography, X-Ray Genetic Variation Kinetics Ligases/*chemistry/genetics/*metabolism Magnesium/*chemistry/*metabolism Models, Molecular Nucleic Acid Conformation *Protein Interaction Domains and Motifs RNA, Catalytic/*chemistry/genetics/*metabolism Sequence Analysis, DNA}, ISSN = {1469-9001 (Electronic) 1355-8382 (Linking)}, DOI = {10.1261/rna.1912509}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19946040}, year = {2009}, type = {Journal Article} } @article{Balke2014, author = {Balke, D. and Zieten, I. and Strahl, A. and Muller, O. and Muller, S.}, title = {Design and characterization of a twin ribozyme for potential repair of a deletion mutation within the oncogenic CTNNB1-DeltaS45 mRNA}, journal = {ChemMedChem}, volume = {9}, number = {9}, pages = {2128-37}, note = {Balke, Darko Zieten, Irene Strahl, Anne Muller, Oliver Muller, Sabine eng Research Support, Non-U.S. Gov't Germany 2014/08/13 06:00 ChemMedChem. 2014 Sep;9(9):2128-37. doi: 10.1002/cmdc.201402166. Epub 2014 Aug 11.}, abstract = {RNA repair is an emerging strategy for gene therapy. Conventional gene therapy typically relies on the addition of the corrected DNA sequence of a defective gene to restore gene function. As an additional option, RNA repair allows alteration of the sequence of endogenous messenger RNAs (mRNAs). mRNA sequence alteration is either facilitated by intracellular spliceosome machinery or by the intrinsic catalytic activity of trans-acting ribozymes. Previously we developed twin ribozymes, derived from the hairpin ribozyme, by tandem duplication and demonstrated their potential for patchwise RNA repair. Herein we describe the development of such a twin ribozyme for potential repair of a deletion mutation in the oncogenic CTNNB1-DeltaS45 mRNA. We demonstrate that hairpin ribozyme units within the twin ribozyme can be adapted to efficiently cleave/ligate non-consensus substrates by introduction of compensatory mutations in the ribozyme. Thus, we show the twin ribozyme mediated repair of truncated CTNNB1 transcripts (up to 1000 nt length). Repair of the entire CTNNB1-DeltaS45 mRNA, although apparently possible in general, is hampered in vitro by the secondary structure of the transcript.}, keywords = {Base Sequence DNA Repair/*drug effects Drug Design *Gene Deletion Kinetics Molecular Sequence Data Mutation/*drug effects RNA Cleavage/drug effects RNA, Catalytic/*chemical synthesis/*pharmacology RNA, Messenger/*drug effects beta Catenin/*drug effects/genetics}, ISSN = {1860-7187 (Electronic) 1860-7179 (Linking)}, DOI = {10.1002/cmdc.201402166}, url = {http://www.ncbi.nlm.nih.gov/pubmed/25112518}, year = {2014}, type = {Journal Article} } @article{Bartel1993, author = {Bartel, D. P. and Szostak, J. W.}, title = {Isolation of new ribozymes from a large pool of random sequences [see comment]}, journal = {Science}, volume = {261}, number = {5127}, pages = {1411-8}, note = {Bartel, D P Szostak, J W eng Comment Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. 1993/09/10 Science. 1993 Sep 10;261(5127):1411-8.}, abstract = {An iterative in vitro selection procedure was used to isolate a new class of catalytic RNAs (ribozymes) from a large pool of random-sequence RNA molecules. These ribozymes ligate two RNA molecules that are aligned on a template by catalyzing the attack of a 3'-hydroxyl on an adjacent 5'-triphosphate--a reaction similar to that employed by the familiar protein enzymes that synthesize RNA. The corresponding uncatalyzed reaction also yields a 3',5'-phosphodiester bond. In vitro evolution of the population of new ribozymes led to improvement of the average ligation activity and the emergence of ribozymes with reaction rates 7 million times faster than the uncatalyzed reaction rate.}, keywords = {Base Sequence Biological Evolution Catalysis Kinetics Magnesium/metabolism Molecular Sequence Data Mutation Oligoribonucleotides/metabolism RNA/*metabolism RNA Ligase (ATP)/chemistry/isolation & purification/metabolism RNA, Catalytic/chemistry/*isolation & purification/metabolism Temperature Templates, Genetic NASA Discipline Exobiology Non-NASA Center}, ISSN = {0036-8075 (Print) 0036-8075 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/7690155}, year = {1993}, type = {Journal Article} } @misc{Bartnicki2014, author = {BARTNICKI, F. and Kowalska, E. and PELS, K. and STRZAŁKA, W.}, title = {Dna aptamers binding the histidine tag and their application}, publisher = {Google Patents}, url = {https://www.google.com/patents/WO2014185802A1?cl=en}, year = {2014}, type = {Generic} } @article{Beaudry1992, author = {Beaudry, A. A. and Joyce, G. F.}, title = {Directed evolution of an RNA enzyme}, journal = {Science}, volume = {257}, number = {5070}, pages = {635-41}, note = {Beaudry, A A Joyce, G F eng AI30882/AI/NIAID NIH HHS/ Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S. 1992/07/31 Science. 1992 Jul 31;257(5070):635-41.}, abstract = {An in vitro evolution procedure was used to obtain RNA enzymes with a particular catalytic function. A population of 10(13) variants of the Tetrahymena ribozyme, a group I ribozyme that catalyzes sequence-specific cleavage of RNA via a phosphoester transfer mechanism, was generated. This enzyme has a limited ability to cleave DNA under conditions of high temperature or high MgCl2 concentration, or both. A selection constraint was imposed on the population of ribozyme variants such that only those individuals that carried out DNA cleavage under physiologic conditions were amplified to produce "progeny" ribozymes. Mutations were introduced during amplification to maintain heterogeneity in the population. This process was repeated for ten successive generations, resulting in enhanced (100 times) DNA cleavage activity.}, keywords = {Animals Base Composition Base Sequence Catalysis DNA, Single-Stranded/metabolism Genotype Hot Temperature Magnesium Chloride/pharmacology Molecular Sequence Data Mutagenesis Mutagenesis, Site-Directed Phenotype Polymerase Chain Reaction RNA, Catalytic/genetics/*metabolism Substrate Specificity Tetrahymena thermophila/*genetics NASA Discipline Exobiology Non-NASA Center}, ISSN = {0036-8075 (Print) 0036-8075 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/1496376}, year = {1992}, type = {Journal Article} } @article{Bock1992, author = {Bock, L. C. and Griffin, L. C. and Latham, J. A. and Vermaas, E. H. and Toole, J. J.}, title = {Selection of single-stranded DNA molecules that bind and inhibit human thrombin}, journal = {Nature}, volume = {355}, number = {6360}, pages = {564-6}, note = {Bock, L C Griffin, L C Latham, J A Vermaas, E H Toole, J J eng ENGLAND 1992/02/06 Nature. 1992 Feb 6;355(6360):564-6.}, abstract = {Aptamers are double-stranded DNA or single-stranded RNA molecules that bind specific molecular targets. Large randomly generated populations can be enriched in aptamers by in vitro selection and polymerase chain reaction. But so far single-stranded DNA has not been investigated for aptamer properties, nor has a target protein been considered that does not interact physiologically with nucleic acid. Here we describe the isolation of single-stranded DNA aptamers to the protease thrombin of the blood coagulation cascade and report binding affinities in the range 25-200 nM. Sequence data from 32 thrombin aptamers, selected from a pool of DNA containing 60 nucleotides of random sequence, displayed a highly conserved 14-17-base region. Several of these aptamers at nanomolar concentrations inhibited thrombin-catalysed fibrin-clot formation in vitro using either purified fibrinogen or human plasma.}, keywords = {Base Sequence Blood Coagulation/drug effects Cloning, Molecular DNA, Single-Stranded/*isolation & purification Dose-Response Relationship, Drug Humans In Vitro Techniques Molecular Sequence Data Polymerase Chain Reaction Sequence Homology, Nucleic Acid Thrombin/*antagonists & inhibitors Thrombin Time}, ISSN = {0028-0836 (Print) 0028-0836 (Linking)}, DOI = {10.1038/355564a0}, url = {http://www.ncbi.nlm.nih.gov/pubmed/1741036}, year = {1992}, type = {Journal Article} } @article{Breaker1994, author = {Breaker, R. R. and Joyce, G. F.}, title = {A DNA enzyme that cleaves RNA}, journal = {Chem Biol}, volume = {1}, number = {4}, pages = {223-9}, note = {Breaker, R R Joyce, G F eng Research Support, U.S. Gov't, Non-P.H.S. ENGLAND 1994/12/01 00:00 Chem Biol. 1994 Dec;1(4):223-9.}, abstract = {BACKGROUND: Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology. RESULTS: Beginning with a population of 10(14) DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb(2+)-dependent cleavage of a target ribonucleoside 3'-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min-1. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min-1. This rate is about 10(5)-fold increased compared to the uncatalyzed reaction. CONCLUSIONS: Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb(2+)-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.}, keywords = {Catalysis DNA/*metabolism Kinetics Lead/chemistry RNA/*metabolism RNA, Catalytic/*metabolism}, ISSN = {1074-5521 (Print) 1074-5521 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/9383394}, year = {1994}, type = {Journal Article} } @article{Buzayan1986, author = {Buzayan, J. M. and Gerlach, W. L. and Bruening, G.}, title = {Nonenzymatic Cleavage and Ligation of Rnas Complementary to a Plant-Virus Satellite Rna}, journal = {Nature}, volume = {323}, number = {6086}, pages = {349-353}, note = {E1371 Times Cited:285 Cited References Count:20}, ISSN = {0028-0836}, DOI = {DOI 10.1038/323349a0}, url = {://WOS:A1986E137100051}, year = {1986}, type = {Journal Article} } @article{Cech1981, author = {Cech, T. R. and Zaug, A. J. and Grabowski, P. J.}, title = {In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence}, journal = {Cell}, volume = {27}, number = {3 Pt 2}, pages = {487-96}, note = {Cech, T R Zaug, A J Grabowski, P J eng Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. 1981/12/01 Cell. 1981 Dec;27(3 Pt 2):487-96.}, abstract = {In previous studies of transcription and splicing of the ribosomal RNA precursor in isolated Tetrahymena nuclei, we found that the intervening sequence (IVS) was excised as a unique linear RNA molecule and was subsequently cyclized. In the present work, transcription at low monovalent cation concentration is found to inhibit splicing and to lead to the accumulation of a splicing intermediate. This intermediate contains splicing activity that either is tightly bound to the RNA or is part of the RNA molecule itself. The intermediate is able to complete the excision of the IVS when it is incubated with a monovalent cation (75 mM (NH4)2SO4), a divalent cation (5-10 mM MgCl2) and a guanosine compound (1 microM GTP, GDP, GMP or guanosine). ATP, UTP, CTP and guanosine compounds without 2' and 3' hydroxyl groups are inactive in causing excision of the IVS. Accurate excision of the IVS, cyclization of the IVS and (apparently) ligation of the 26S rRNA sequences bordering the IVS all take place under these conditions, suggesting that a single activity is responsible for all three reactions. During excision of the IVS, the 3' hydroxyl of the guanosine moiety becomes linked to the 5' end of the IVS RNA via a normal phosphodiester bond. When GTP is used to drive the reaction, it is added intact without hydrolysis. Based on these results, we propose that Tetrahymena pre-rRNA splicing occurs by a phosphoester transferase mechanism. According to this model, the guanosine cofactor provides the free 3' hydroxyl necessary to initiate a series of three transfers that results in splicing of the pre-rRNA and cyclization of the excised IVS.}, keywords = {Animals Base Sequence Guanine Nucleotides/metabolism Introns Models, Biological Molecular Sequence Data Nucleic Acid Precursors/genetics/metabolism *RNA Splicing RNA, Ribosomal/genetics/metabolism Tetrahymena thermophila/genetics/*metabolism}, ISSN = {0092-8674 (Print) 0092-8674 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/6101203}, year = {1981}, type = {Journal Article} } @article{Crick1958, author = {Crick, F. H.}, title = {On protein synthesis}, journal = {Symp Soc Exp Biol}, volume = {12}, pages = {138-63}, note = {CRICK, F H eng Not Available 1958/01/01 Symp Soc Exp Biol. 1958;12:138-63.}, keywords = {Proteins/*metabolism *PROTEINS/metabolism}, ISSN = {0081-1386 (Print) 0081-1386 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/13580867}, year = {1958}, type = {Journal Article} } @article{Ellington1990, author = {Ellington, A. D. and Szostak, J. W.}, title = {In vitro selection of RNA molecules that bind specific ligands}, journal = {Nature}, volume = {346}, number = {6287}, pages = {818-22}, note = {Ellington, A D Szostak, J W eng Research Support, Non-U.S. Gov't ENGLAND 1990/08/30 Nature. 1990 Aug 30;346(6287):818-22.}, abstract = {Subpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules. Roughly one in 10(10) random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands.}, keywords = {Base Sequence Binding Sites Chromatography, Affinity Cloning, Molecular Coloring Agents/*metabolism DNA/chemical synthesis Ligands Molecular Sequence Data Molecular Structure Nucleic Acid Conformation Polymerase Chain Reaction RNA/biosynthesis/*isolation & purification/metabolism Templates, Genetic Transcription, Genetic Triazines/metabolism}, ISSN = {0028-0836 (Print) 0028-0836 (Linking)}, DOI = {10.1038/346818a0}, url = {http://www.ncbi.nlm.nih.gov/pubmed/1697402}, year = {1990}, type = {Journal Article} } @article{Forster1987, author = {Forster, A. C. and Symons, R. H.}, title = {Self-Cleavage of Virusoid Rna Is Performed by the Proposed 55-Nucleotide Active-Site}, journal = {Cell}, volume = {50}, number = {1}, pages = {9-16}, note = {J1550 Times Cited:236 Cited References Count:23}, ISSN = {0092-8674}, DOI = {Doi 10.1016/0092-8674(87)90657-X}, url = {://WOS:A1987J155000004}, year = {1987}, type = {Journal Article} } @article{Grate1999, author = {Grate, D. and Wilson, C.}, title = {Laser-mediated, site-specific inactivation of RNA transcripts}, journal = {Proc Natl Acad Sci U S A}, volume = {96}, number = {11}, pages = {6131-6}, ISSN = {0027-8424 (Print) 0027-8424 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/10339553}, year = {1999}, type = {Journal Article} } @article{Hershey1952, author = {Hershey, A. D. and Chase, M.}, title = {Independent functions of viral protein and nucleic acid in growth of bacteriophage}, journal = {J Gen Physiol}, volume = {36}, number = {1}, pages = {39-56}, note = {HERSHEY, A D CHASE, M eng Not Available 1952/05/01 J Gen Physiol. 1952 May;36(1):39-56.}, abstract = {1. Osmotic shock disrupts particles of phage T2 into material containing nearly all the phage sulfur in a form precipitable by antiphage serum, and capable of specific adsorption to bacteria. It releases into solution nearly all the phage DNA in a form not precipitable by antiserum and not adsorbable to bacteria. The sulfur-containing protein of the phage particle evidently makes up a membrane that protects the phage DNA from DNase, comprises the sole or principal antigenic material, and is responsible for attachment of the virus to bacteria. 2. Adsorption of T2 to heat-killed bacteria, and heating or alternate freezing and thawing of infected cells, sensitize the DNA of the adsorbed phage to DNase. These treatments have little or no sensitizing effect on unadsorbed phage. Neither heating nor freezing and thawing releases the phage DNA from infected cells, although other cell constituents can be extracted by these methods. These facts suggest that the phage DNA forms part of an organized intracellular structure throughout the period of phage growth. 3. Adsorption of phage T2 to bacterial debris causes part of the phage DNA to appear in solution, leaving the phage sulfur attached to the debris. Another part of the phage DNA, corresponding roughly to the remaining half of the DNA of the inactivated phage, remains attached to the debris but can be separated from it by DNase. Phage T4 behaves similarly, although the two phages can be shown to attach to different combining sites. The inactivation of phage by bacterial debris is evidently accompanied by the rupture of the viral membrane. 4. Suspensions of infected cells agitated in a Waring blendor release 75 per cent of the phage sulfur and only 15 per cent of the phage phosphorus to the solution as a result of the applied shearing force. The cells remain capable of yielding phage progeny. 5. The facts stated show that most of the phage sulfur remains at the cell surface and most of the phage DNA enters the cell on infection. Whether sulfur-free material other than DNA enters the cell has not been determined. The properties of the sulfur-containing residue identify it as essentially unchanged membranes of the phage particles. All types of evidence show that the passage of phage DNA into the cell occurs in non-nutrient medium under conditions in which other known steps in viral growth do not occur. 6. The phage progeny yielded by bacteria infected with phage labeled with radioactive sulfur contain less than 1 per cent of the parental radioactivity. The progeny of phage particles labeled with radioactive phosphorus contain 30 per cent or more of the parental phosphorus. 7. Phage inactivated by dilute formaldehyde is capable of adsorbing to bacteria, but does not release its DNA to the cell. This shows that the interaction between phage and bacterium resulting in release of the phage DNA from its protective membrane depends on labile components of the phage particle. By contrast, the components of the bacterium essential to this interaction are remarkably stable. The nature of the interaction is otherwise unknown. 8. The sulfur-containing protein of resting phage particles is confined to a protective coat that is responsible for the adsorption to bacteria, and functions as an instrument for the injection of the phage DNA into the cell. This protein probably has no function in the growth of intracellular phage. The DNA has some function. Further chemical inferences should not be drawn from the experiments presented.}, keywords = {Bacteriophages/*metabolism Nucleic Acids/*metabolism}, ISSN = {0022-1295 (Print) 0022-1295 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/12981234}, year = {1952}, type = {Journal Article} } @article{Himo2005, author = {Himo, F. and Lovell, T. and Hilgraf, R. and Rostovtsev, V. V. and Noodleman, L. and Sharpless, K. B. and Fokin, V. V.}, title = {Copper(I)-catalyzed synthesis of azoles. DFT study predicts unprecedented reactivity and intermediates}, journal = {J Am Chem Soc}, volume = {127}, number = {1}, pages = {210-6}, note = {Himo, Fahmi Lovell, Timothy Hilgraf, Robert Rostovtsev, Vsevolod V Noodleman, Louis Sharpless, K Barry Fokin, Valery V eng GM28384/GM/NIGMS NIH HHS/ Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S. 2005/01/06 09:00 J Am Chem Soc. 2005 Jan 12;127(1):210-6.}, abstract = {Huisgen's 1,3-dipolar cycloadditions become nonconcerted when copper(I) acetylides react with azides and nitrile oxides, providing ready access to 1,4-disubstituted 1,2,3-triazoles and 3,4-disubstituted isoxazoles, respectively. The process is highly reliable and exhibits an unusually wide scope with respect to both components. Computational studies revealed a stepwise mechanism involving unprecedented metallacycle intermediates, which appear to be common for a variety of dipoles.}, keywords = {Catalysis Cations, Monovalent Copper/*chemistry Isoxazoles/*chemical synthesis Models, Molecular Nitriles/chemistry Thermodynamics Triazoles/*chemical synthesis}, ISSN = {0002-7863 (Print) 0002-7863 (Linking)}, DOI = {10.1021/ja0471525}, url = {http://www.ncbi.nlm.nih.gov/pubmed/15631470}, year = {2005}, type = {Journal Article} } @article{Jenison1994, author = {Jenison, R. D. and Gill, S. C. and Pardi, A. and Polisky, B.}, title = {High-resolution molecular discrimination by RNA}, journal = {Science}, volume = {263}, number = {5152}, pages = {1425-9}, note = {Jenison, R D Gill, S C Pardi, A Polisky, B eng AI01051/AI/NIAID NIH HHS/ AI33098/AI/NIAID NIH HHS/ RR03283/RR/NCRR NIH HHS/ Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. 1994/03/11 Science. 1994 Mar 11;263(5152):1425-9.}, abstract = {Species of RNA that bind with high affinity and specificity to the bronchodilator theophylline were identified by selection from an oligonucleotide library. One RNA molecule binds to theophylline with a dissociation constant Kd of 0.1 microM. This binding affinity is 10,000-fold greater than the RNA molecule's affinity for caffeine, which differs from theophylline only by a methyl group at nitrogen atom N-7. Analysis by nuclear magnetic resonance indicates that this RNA molecule undergoes a significant change in its conformation or dynamics upon theophylline binding. Binding studies of compounds chemically related to theophylline have revealed structural features required for the observed binding specificity. These results demonstrate the ability of RNA molecules to exhibit an extremely high degree of ligand recognition and discrimination.}, keywords = {Base Sequence Binding Sites Binding, Competitive DNA, Complementary/chemistry Hydrogen Bonding Magnetic Resonance Spectroscopy Molecular Sequence Data Molecular Structure Nucleic Acid Conformation RNA/chemistry/*metabolism Sequence Analysis, DNA Theophylline/chemistry/*metabolism Xanthines/chemistry/metabolism}, ISSN = {0036-8075 (Print) 0036-8075 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/7510417}, year = {1994}, type = {Journal Article} } @article{Joyce1989, author = {Joyce, G. F.}, title = {Amplification, mutation and selection of catalytic RNA}, journal = {Gene}, volume = {82}, number = {1}, pages = {83-7}, note = {Joyce, G F eng NETHERLANDS 1989/10/15 Gene. 1989 Oct 15;82(1):83-7.}, abstract = {RNA, by virtue of its genotypic and phenotypic properties, is a suitable substrate for molecular evolution in the laboratory. We have developed techniques for the rapid amplification, mutation and selection of catalytic RNA. By combining these techniques in an iterative fashion, we are attempting to construct an RNA-based evolving system. Such a system could be used to explore the catalytic potential of RNA.}, keywords = {Biological Evolution Catalysis Gene Amplification *Genetic Techniques Mutation Nucleic Acid Conformation RNA, Catalytic RNA, Ribosomal/*genetics Selection, Genetic}, ISSN = {0378-1119 (Print) 0378-1119 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/2684778}, year = {1989}, type = {Journal Article} } @article{Kolb2001, author = {Kolb, H. C. and Finn, M. G. and Sharpless, K. B.}, title = {Click Chemistry: Diverse Chemical Function from a Few Good Reactions}, journal = {Angew Chem Int Ed Engl}, volume = {40}, number = {11}, pages = {2004-2021}, note = {Kolb, Hartmuth C. Finn, M. G. Sharpless, K. Barry ENG 2001/07/04 10:00 Angew Chem Int Ed Engl. 2001 Jun 1;40(11):2004-2021.}, abstract = {Examination of nature's favorite molecules reveals a striking preference for making carbon-heteroatom bonds over carbon-carbon bonds-surely no surprise given that carbon dioxide is nature's starting material and that most reactions are performed in water. Nucleic acids, proteins, and polysaccharides are condensation polymers of small subunits stitched together by carbon-heteroatom bonds. Even the 35 or so building blocks from which these crucial molecules are made each contain, at most, six contiguous C-C bonds, except for the three aromatic amino acids. Taking our cue from nature's approach, we address here the development of a set of powerful, highly reliable, and selective reactions for the rapid synthesis of useful new compounds and combinatorial libraries through heteroatom links (C-X-C), an approach we call "click chemistry". Click chemistry is at once defined, enabled, and constrained by a handful of nearly perfect "spring-loaded" reactions. The stringent criteria for a process to earn click chemistry status are described along with examples of the molecular frameworks that are easily made using this spartan, but powerful, synthetic strategy.}, ISSN = {1521-3773 (Electronic) 1433-7851 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/11433435}, year = {2001}, type = {Journal Article} } @article{Kruger1982, author = {Kruger, K. and Grabowski, P. J. and Zaug, A. J. and Sands, J. and Gottschling, D. E. and Cech, T. R.}, title = {Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena}, journal = {Cell}, volume = {31}, number = {1}, pages = {147-57}, note = {Kruger, K Grabowski, P J Zaug, A J Sands, J Gottschling, D E Cech, T R eng Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. 1982/11/01 Cell. 1982 Nov;31(1):147-57.}, abstract = {In the macronuclear rRNA genes of Tetrahymena thermophila, a 413 bp intervening sequence (IVS) interrupts the 26S rRNA-coding region. A restriction fragment of the rDNA containing the IVS and portions of the adjacent rRNA sequences (exons) was inserted downstream from the lac UV5 promoter in a recombinant plasmid. Transcription of this template by purified Escherichia coli RNA polymerase in vitro produced a shortened version of the pre-rRNA, which was then deproteinized. When incubated with monovalent and divalent cations and a guanosine factor, this RNA underwent splicing. The reactions that were characterized included the precise excision of the IVS, attachment of guanosine to the 5' end of the IVS, covalent cyclization of the IVS and ligation of the exons. We conclude that splicing activity is intrinsic to the structure of the RNA, and that enzymes, small nuclear RNAs and folding of the pre-rRNA into an RNP are unnecessary for these reactions. We propose that the IVS portion of the RNA has several enzyme-like properties that enable it to break and reform phosphodiester bonds. The finding of autocatalytic rearrangements of RNA molecules has implications for the mechanism and the evolution of other reactions that involve RNA.}, keywords = {Animals Base Sequence DNA Restriction Enzymes DNA Transposable Elements *Genes Lac Operon Nucleic Acid Hybridization Plasmids RNA, Ribosomal/*genetics Tetrahymena/*genetics Transcription, Genetic}, ISSN = {0092-8674 (Print) 0092-8674 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/6297745}, year = {1982}, type = {Journal Article} } @article{Lam2009, author = {Lam, B. J. and Joyce, G. F.}, title = {Autocatalytic aptazymes enable ligand-dependent exponential amplification of RNA}, journal = {Nat Biotechnol}, volume = {27}, number = {3}, pages = {288-92}, note = {Lam, Bianca J Joyce, Gerald F eng 5F32GM078691/GM/NIGMS NIH HHS/ F32 GM078691-03/GM/NIGMS NIH HHS/ R01 GM065130/GM/NIGMS NIH HHS/ R01 GM065130-07/GM/NIGMS NIH HHS/ R01GM065130/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't 2009/02/24 09:00 Nat Biotechnol. 2009 Mar;27(3):288-92. doi: 10.1038/nbt.1528. Epub 2009 Feb 22.}, abstract = {RNA enzymes have been developed that undergo self-sustained replication at a constant temperature in the absence of proteins. These RNA molecules amplify exponentially through a cross-replicative process, whereby two enzymes catalyze each other's synthesis by joining component oligonucleotides. Other RNA enzymes have been made to operate in a ligand-dependent manner by combining a catalytic domain with a ligand-binding domain (aptamer) to produce an 'aptazyme'. The principle of ligand-dependent RNA catalysis has now been extended to the cross-replicating RNA enzymes so that exponential amplification occurs in the presence, but not the absence, of the cognate ligand. The exponential growth rate of the RNA depends on the concentration of the ligand, allowing one to determine the concentration of ligand in a sample. This process is analogous to quantitative PCR (qPCR) but can be generalized to a wide variety of targets, including proteins and small molecules that are relevant to medical diagnostics and environmental monitoring.}, keywords = {Aptamers, Nucleotide/*metabolism Biocatalysis Kinetics Ligands Nucleic Acid Conformation Polynucleotide Ligases/*metabolism RNA/*metabolism RNA Ligase (ATP)/*metabolism RNA, Catalytic/*metabolism}, ISSN = {1546-1696 (Electronic) 1087-0156 (Linking)}, DOI = {10.1038/nbt.1528}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19234448}, year = {2009}, type = {Journal Article} } @article{Levy2005, author = {Levy, M. and Griswold, K. E. and Ellington, A. D.}, title = {Direct selection of trans-acting ligase ribozymes by in vitro compartmentalization}, journal = {RNA}, volume = {11}, number = {10}, pages = {1555-62}, note = {Levy, Matthew Griswold, Karl E Ellington, Andrew D eng 8R01 EB002043/EB/NIBIB NIH HHS/ Comparative Study Research Support, N.I.H., Extramural Research Support, U.S. Gov't, P.H.S. New York, N.Y. 2005/09/01 09:00 RNA. 2005 Oct;11(10):1555-62. Epub 2005 Aug 30.}, abstract = {We have used a compartmentalized in vitro selection method to directly select for ligase ribozymes that are capable of acting on and turning over separable oligonucleotide substrates. Starting from a degenerate pool, we selected a trans-acting variant of the Bartel class I ligase which statistically may have been the only active variant in the starting pool. The isolation of this sequence from the population suggests that this selection method is extremely robust at selecting optimal ribozymes and should, therefore, prove useful for the selection and optimization of other trans-acting nucleic acid catalysts capable of multiple turnover catalysis.}, keywords = {Base Pairing Base Sequence Cloning, Molecular DNA/chemistry Emulsions Flow Cytometry Fluorescein Fluorescent Dyes Genetic Variation Hydrazines In Vitro Techniques Kinetics Ligases/*metabolism Microspheres Models, Chemical Nucleic Acid Amplification Techniques Nucleic Acid Conformation RNA, Catalytic/*chemistry/genetics/*isolation & purification/metabolism Sequence Analysis, RNA Substrate Specificity Transcription, Genetic}, ISSN = {1355-8382 (Print) 1355-8382 (Linking)}, DOI = {10.1261/rna.2121705}, url = {http://www.ncbi.nlm.nih.gov/pubmed/16131588}, year = {2005}, type = {Journal Article} } @article{Levy2005, author = {Levy, M. and Griswold, K. E. and Ellington, A. D.}, title = {Direct selection of trans-acting ligase ribozymes by in vitro compartmentalization}, journal = {RNA}, volume = {11}, number = {10}, pages = {1555-62}, note = {Levy, Matthew Griswold, Karl E Ellington, Andrew D eng 8R01 EB002043/EB/NIBIB NIH HHS/ Comparative Study Research Support, N.I.H., Extramural Research Support, U.S. Gov't, P.H.S. New York, N.Y. 2005/09/01 09:00 RNA. 2005 Oct;11(10):1555-62. Epub 2005 Aug 30.}, abstract = {We have used a compartmentalized in vitro selection method to directly select for ligase ribozymes that are capable of acting on and turning over separable oligonucleotide substrates. Starting from a degenerate pool, we selected a trans-acting variant of the Bartel class I ligase which statistically may have been the only active variant in the starting pool. The isolation of this sequence from the population suggests that this selection method is extremely robust at selecting optimal ribozymes and should, therefore, prove useful for the selection and optimization of other trans-acting nucleic acid catalysts capable of multiple turnover catalysis.}, keywords = {Base Pairing Base Sequence Cloning, Molecular DNA/chemistry Emulsions Flow Cytometry Fluorescein Fluorescent Dyes Genetic Variation Hydrazines In Vitro Techniques Kinetics Ligases/*metabolism Microspheres Models, Chemical Nucleic Acid Amplification Techniques Nucleic Acid Conformation RNA, Catalytic/*chemistry/genetics/*isolation & purification/metabolism Sequence Analysis, RNA Substrate Specificity Transcription, Genetic}, ISSN = {1355-8382 (Print) 1355-8382 (Linking)}, DOI = {10.1261/rna.2121705}, url = {http://www.ncbi.nlm.nih.gov/pubmed/16131588}, year = {2005}, type = {Journal Article} } @article{Lincoln2009, author = {Lincoln, T. A. and Joyce, G. F.}, title = {Self-sustained replication of an RNA enzyme}, journal = {Science}, volume = {323}, number = {5918}, pages = {1229-32}, note = {Lincoln, Tracey A Joyce, Gerald F eng R01 GM065130/GM/NIGMS NIH HHS/ R01 GM065130-07/GM/NIGMS NIH HHS/ R01GM065130/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. 2009/01/10 09:00 Science. 2009 Feb 27;323(5918):1229-32. doi: 10.1126/science.1167856. Epub 2009 Jan 8.}, abstract = {An RNA enzyme that catalyzes the RNA-templated joining of RNA was converted to a format whereby two enzymes catalyze each other's synthesis from a total of four oligonucleotide substrates. These cross-replicating RNA enzymes undergo self-sustained exponential amplification in the absence of proteins or other biological materials. Amplification occurs with a doubling time of about 1 hour and can be continued indefinitely. Populations of various cross-replicating enzymes were constructed and allowed to compete for a common pool of substrates, during which recombinant replicators arose and grew to dominate the population. These replicating RNA enzymes can serve as an experimental model of a genetic system. Many such model systems could be constructed, allowing different selective outcomes to be related to the underlying properties of the genetic system.}, keywords = {Base Pairing Biocatalysis Directed Molecular Evolution Kinetics Nucleic Acid Conformation Oligonucleotides/*metabolism Polynucleotide Ligases/*chemistry/metabolism RNA, Catalytic/chemistry/*metabolism}, ISSN = {1095-9203 (Electronic) 0036-8075 (Linking)}, DOI = {10.1126/science.1167856}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19131595}, year = {2009}, type = {Journal Article} } @article{Lorenz2015, author = {Lorenz, D. A. and Song, J. M. and Garner, A. L.}, title = {High-throughput platform assay technology for the discovery of pre-microrna-selective small molecule probes}, journal = {Bioconjug Chem}, volume = {26}, number = {1}, pages = {19-23}, note = {Lorenz, Daniel A Song, James M Garner, Amanda L eng Research Support, Non-U.S. Gov't 2014/12/17 06:00 Bioconjug Chem. 2015 Jan 21;26(1):19-23. doi: 10.1021/bc500544v. Epub 2014 Dec 17.}, abstract = {MicroRNAs (miRNA) play critical roles in human development and disease. As such, the targeting of miRNAs is considered attractive as a novel therapeutic strategy. A major bottleneck toward this goal, however, has been the identification of small molecule probes that are specific for select RNAs and methods that will facilitate such discovery efforts. Using pre-microRNAs as proof-of-concept, herein we report a conceptually new and innovative approach for assaying RNA-small molecule interactions. Through this platform assay technology, which we term catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a method that can be implemented in high throughput, is virtually free of false readouts, and is general for all nucleic acids. Through cat-ELCCA, we envision the discovery of selective small molecule ligands for disease-relevant miRNAs to promote the field of RNA-targeted drug discovery and further our understanding of the role of miRNAs in cellular biology.}, keywords = {Biocatalysis Drug Discovery/*methods High-Throughput Screening Assays/*methods MicroRNAs/*metabolism Molecular Probes/*chemistry/*metabolism Ribonuclease III/metabolism Substrate Specificity}, ISSN = {1520-4812 (Electronic) 1043-1802 (Linking)}, DOI = {10.1021/bc500544v}, url = {http://www.ncbi.nlm.nih.gov/pubmed/25506628}, year = {2015}, type = {Journal Article} } @article{Matsumura2009, author = {Matsumura, S. and Ohmori, R. and Saito, H. and Ikawa, Y. and Inoue, T.}, title = {Coordinated control of a designed trans-acting ligase ribozyme by a loop-receptor interaction}, journal = {FEBS Lett}, volume = {583}, number = {17}, pages = {2819-26}, note = {Matsumura, Shigeyoshi Ohmori, Rei Saito, Hirohide Ikawa, Yoshiya Inoue, Tan eng Research Support, Non-U.S. Gov't Netherlands 2009/07/28 09:00 FEBS Lett. 2009 Sep 3;583(17):2819-26. doi: 10.1016/j.febslet.2009.07.036. Epub 2009 Jul 23.}, abstract = {We previously developed a synthetic cis-acting RNA ligase ribozyme with 3'-5' joining activity termed "DSL" (designed and selected ligase). DSL was easily transformed into a trans-acting form because of its highly modular architecture. In this study, we investigated the modular properties and turnover capabilities of a trans-acting DSL, tDSL-1/GUAA. tDSL-1/GUAA exhibited remarkably high activity compared with the parental cis-acting DSL, and it attained a high turnover number. Taken together, the results indicate that a loop-receptor interaction plays a significant role in determining the activity of the trans-acting ribozyme and in its ability to perform multiple turnovers of the reaction.}, keywords = {Base Pairing Base Sequence Molecular Sequence Data *Nucleic Acid Conformation RNA Ligase (ATP)/*chemistry/genetics/*metabolism RNA, Catalytic/*chemistry/genetics/*metabolism Substrate Specificity}, ISSN = {1873-3468 (Electronic) 0014-5793 (Linking)}, DOI = {10.1016/j.febslet.2009.07.036}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19631647}, year = {2009}, type = {Journal Article} } @article{McKay2014, author = {McKay, C. S. and Finn, M. G.}, title = {Click chemistry in complex mixtures: bioorthogonal bioconjugation}, journal = {Chem Biol}, volume = {21}, number = {9}, pages = {1075-101}, note = {McKay, Craig S Finn, M G eng R01 GM101421/GM/NIGMS NIH HHS/ Review 2014/09/23 06:00 Chem Biol. 2014 Sep 18;21(9):1075-101. doi: 10.1016/j.chembiol.2014.09.002.}, abstract = {The selective chemical modification of biological molecules drives a good portion of modern drug development and fundamental biological research. While a few early examples of reactions that engage amine and thiol groups on proteins helped establish the value of such processes, the development of reactions that avoid most biological molecules so as to achieve selectivity in desired bond-forming events has revolutionized the field. We provide an update on recent developments in bioorthogonal chemistry that highlights key advances in reaction rates, biocompatibility, and applications. While not exhaustive, we hope this summary allows the reader to appreciate the rich continuing development of good chemistry that operates in the biological setting.}, keywords = {Aldehydes/chemistry Amines/chemistry Amino Acids/chemistry *Click Chemistry Hydroxides/chemistry Ketones/chemistry Organometallic Compounds/chemistry Prodrugs/chemistry/metabolism Proteins/*chemistry/metabolism}, ISSN = {1879-1301 (Electronic) 1074-5521 (Linking)}, DOI = {10.1016/j.chembiol.2014.09.002}, url = {http://www.ncbi.nlm.nih.gov/pubmed/25237856}, year = {2014}, type = {Journal Article} } @article{Meyer2014, author = {Meyer, M. and Masquida, B.}, title = {cis-Acting 5' hammerhead ribozyme optimization for in vitro transcription of highly structured RNAs}, journal = {Methods Mol Biol}, volume = {1086}, pages = {21-40}, note = {Meyer, Melanie Masquida, Benoit eng Research Support, Non-U.S. Gov't 2013/10/19 06:00 Methods Mol Biol. 2014;1086:21-40. doi: 10.1007/978-1-62703-667-2_2.}, abstract = {RNA-mediated biological processes usually require precise definition of 5' and 3' ends. RNA ends obtained by in vitro transcription using T7 RNA polymerase are often heterogeneous in length and sequence. An efficient strategy to overcome these drawbacks consists of inserting an RNA with known boundaries in between two ribozymes, usually a 5' hammerhead and a 3' hepatitis delta virus ribozymes, that cleave off the desired RNA. In practice, folding of the three RNAs challenges each other, potentially preventing thorough processing. Folding and cleavage of the 5' hammerhead ribozyme relies on a sequence of nucleotides belonging to the central RNA making it more sensitive than the usual 3' hepatitis delta virus ribozyme. The intrinsic stability of the central RNA may thus prevent correct processing of the full transcript. Here, we present a method in which incorporation of a full-length hammerhead ribozyme with a specific tertiary interaction prevents alternative folding with the lariat capping GIR1 ribozyme and enables complete cleavage in the course of the transcription. This strategy may be transposable for in vitro transcription of any highly structured RNA.}, keywords = {Animals Base Sequence Blotting, Northern/methods Cloning, Molecular/methods Computer Simulation Models, Molecular Molecular Sequence Data Nucleic Acid Conformation RNA/chemistry/genetics/*metabolism RNA, Catalytic/chemistry/genetics/*metabolism RNA, Helminth/chemistry/genetics/*metabolism Recombinant Fusion Proteins/chemistry/genetics/metabolism Schistosoma/chemistry/*enzymology/genetics/metabolism Software Transcription, Genetic}, ISSN = {1940-6029 (Electronic) 1064-3745 (Linking)}, DOI = {10.1007/978-1-62703-667-2_2}, url = {http://www.ncbi.nlm.nih.gov/pubmed/24136596}, year = {2014}, type = {Journal Article} } @article{Ohmori2011, author = {Ohmori, R. and Saito, H. and Ikawa, Y. and Fujita, Y. and Inoue, T.}, title = {Self-replication reactions dependent on tertiary interaction motifs in an RNA ligase ribozyme}, journal = {J Mol Evol}, volume = {73}, number = {3-4}, pages = {221-9}, note = {Ohmori, Rei Saito, Hirohide Ikawa, Yoshiya Fujita, Yoshihiko Inoue, Tan eng Research Support, Non-U.S. Gov't Germany 2011/11/15 06:00 J Mol Evol. 2011 Oct;73(3-4):221-9. doi: 10.1007/s00239-011-9471-2. Epub 2011 Nov 12.}, abstract = {RNA can function both as an informational molecule and as a catalyst in living organisms. This duality is the premise of the RNA world hypothesis. However, one flaw in the hypothesis that RNA was the most essential molecule in primitive life is that no RNA self-replicating system has been found in nature. To verify whether RNA has the potential for self-replication, we constructed a new RNA self-assembling ribozyme that could have conducted an evolvable RNA self-replication reaction. The artificially designed, in vitro selected ligase ribozyme was employed as a prototype for a self-assembling ribozyme. The ribozyme is composed of two RNA fragments (form R1.Z1) that recognize another R1.Z1 molecule as their substrate and perform the high turnover ligation reaction via two RNA tertiary interaction motifs. Furthermore, the substrate recognition of R1.Z1 is tolerant of mutations, generating diversity in the corresponding RNA self-replicating network. Thus, we propose that our system implies the significance of RNA tertiary motifs in the early RNA molecular evolution of the RNA world.}, keywords = {Base Pairing Base Sequence Binding Sites Evolution, Molecular Kinetics Ligases/chemical synthesis/*chemistry Models, Genetic Models, Molecular Molecular Sequence Data *Nucleotide Motifs RNA Folding RNA, Catalytic/chemical synthesis/*chemistry}, ISSN = {1432-1432 (Electronic) 0022-2844 (Linking)}, DOI = {10.1007/s00239-011-9471-2}, url = {http://www.ncbi.nlm.nih.gov/pubmed/22080218}, year = {2011}, type = {Journal Article} } @article{Paige2011, author = {Paige, J. S. and Wu, K. Y. and Jaffrey, S. R.}, title = {RNA mimics of green fluorescent protein}, journal = {Science}, volume = {333}, number = {6042}, pages = {642-6}, note = {Paige, Jeremy S Wu, Karen Y Jaffrey, Samie R eng NS064516/NS/NINDS NIH HHS/ R01 NS064516/NS/NINDS NIH HHS/ R01 NS064516-03/NS/NINDS NIH HHS/ T32 CA062948/CA/NCI NIH HHS/ T32 CA062948-14/CA/NCI NIH HHS/ T32CA062948/CA/NCI NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't 2011/07/30 06:00 Science. 2011 Jul 29;333(6042):642-6. doi: 10.1126/science.1207339.}, abstract = {Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here, we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex, termed Spinach, resembles enhanced GFP and emits a green fluorescence comparable in brightness with fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.}, keywords = {Aptamers, Nucleotide/*chemistry/*metabolism Benzyl Compounds/*chemistry/*metabolism Biomimetics Cell Nucleus/metabolism Cytoplasmic Granules/metabolism Cytosol/metabolism *Fluorescence Green Fluorescent Proteins/*chemistry HEK293 Cells Humans Imidazolines/*chemistry/*metabolism Molecular Mimicry Nucleic Acid Conformation Photobleaching Protein Binding RNA, Untranslated/metabolism SELEX Aptamer Technique Spectrometry, Fluorescence Sucrose/pharmacology}, ISSN = {1095-9203 (Electronic) 0036-8075 (Linking)}, DOI = {10.1126/science.1207339}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21798953}, year = {2011}, type = {Journal Article} } @article{Prody1986, author = {Prody, G. A. and Bakos, J. T. and Buzayan, J. M. and Schneider, I. R. and Bruening, G.}, title = {Autolytic processing of dimeric plant virus satellite RNA}, journal = {Science}, volume = {231}, number = {4745}, pages = {1577-80}, note = {Prody, G A Bakos, J T Buzayan, J M Schneider, I R Bruening, G eng 1986/03/28 00:00 Science. 1986 Mar 28;231(4745):1577-80.}, abstract = {Associated with some plant viruses are small satellite RNA's that depend on the plant virus to provide protective coat protein and presumably at least some of the proteins necessary for satellite RNA replication. Multimeric forms of the satellite RNA of tobacco ringspot virus are probable in vivo precursors of the monomeric satellite RNA. Evidence is presented for the in vitro autolytic processing of dimeric and trimeric forms of this satellite RNA. The reaction generates biologically active monomeric satellite RNA, apparently is reversible to form dimeric RNA from monomeric RNA, and does not require an enzyme for its catalysis.}, ISSN = {0036-8075 (Print) 0036-8075 (Linking)}, DOI = {10.1126/science.231.4745.1577}, url = {http://www.ncbi.nlm.nih.gov/pubmed/17833317}, year = {1986}, type = {Journal Article} } @article{Santoro1997, author = {Santoro, S. W. and Joyce, G. F.}, title = {A general purpose RNA-cleaving DNA enzyme}, journal = {Proc Natl Acad Sci U S A}, volume = {94}, number = {9}, pages = {4262-6}, note = {Santoro, S W Joyce, G F eng Research Support, Non-U.S. Gov't 1997/04/29 Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4262-6.}, abstract = {An in vitro selection procedure was used to develop a DNA enzyme that can be made to cleave almost any targeted RNA substrate under simulated physiological conditions. The enzyme is comprised of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of seven to eight deoxynucleotides each. The RNA substrate is bound through Watson-Crick base pairing and is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. Despite its small size, the DNA enzyme has a catalytic efficiency (kcat/Km) of approximately 10(9) M-1.min-1 under multiple turnover conditions, exceeding that of any other known nucleic acid enzyme. Its activity is dependent on the presence of Mg2+ ion. By changing the sequence of the substrate-recognition domains, the DNA enzyme can be made to target different RNA substrates. In this study, for example, it was directed to cleave synthetic RNAs corresponding to the start codon region of HIV-1 gag/pol, env, vpr, tat, and nef mRNAs.}, keywords = {*DNA, Catalytic DNA, Single-Stranded/*metabolism Genes, Viral *Hiv-1 Oligodeoxyribonucleotides/metabolism Oligoribonucleotides/metabolism RNA, Catalytic/metabolism RNA, Messenger/*metabolism RNA, Viral/*metabolism Selection, Genetic}, ISSN = {0027-8424 (Print) 0027-8424 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/9113977}, year = {1997}, type = {Journal Article} } @article{Santoro1998, author = {Santoro, S. W. and Joyce, G. F.}, title = {Mechanism and utility of an RNA-cleaving DNA enzyme}, journal = {Biochemistry}, volume = {37}, number = {38}, pages = {13330-42}, note = {Santoro, S W Joyce, G F eng AI30882/AI/NIAID NIH HHS/ Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. 1998/09/28 Biochemistry. 1998 Sep 22;37(38):13330-42.}, abstract = {We previously reported the in vitro selection of a general-purpose RNA-cleaving DNA enzyme that exhibits a catalytic efficiency (kcat/KM) exceeding that of any other known nucleic acid enzyme [Santoro, S. W. and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4262-4266]. This enzyme contains approximately 30 deoxynucleotides and can cleave almost any RNA substrate under simulated physiological conditions, recognizing the substrate through two Watson-Crick binding domains. The kinetics of cleavage under conditions of varying pH, choice of divalent metal cofactor, and divalent metal concentration are consistent with a chemical mechanism involving metal-assisted deprotonation of a 2'-hydroxyl of the substrate, leading to substrate cleavage. Kinetic measurements reveal that the enzyme strongly prefers cleavage of the substrate over ligation of the two cleavage products and that the enzyme's catalytic efficiency is limited by the rate of substrate binding. The enzyme displays a high level of substrate specificity, discriminating against RNAs that contain a single base mismatch within either of the two substrate-recognition domains. With appropriate design of the substrate-recognition domains, the enzyme exhibits a potent combination of high substrate sequence specificity and selectivity, high catalytic efficiency, and rapid catalytic turnover.}, keywords = {Base Composition Catalysis Cations, Divalent DNA/*chemistry/metabolism *DNA, Catalytic DNA, Single-Stranded/*metabolism Kinetics Metals/chemistry/metabolism Oligonucleotides/chemistry/metabolism RNA, Catalytic/*chemistry/metabolism Substrate Specificity}, ISSN = {0006-2960 (Print) 0006-2960 (Linking)}, DOI = {10.1021/bi9812221}, url = {http://www.ncbi.nlm.nih.gov/pubmed/9748341}, year = {1998}, type = {Journal Article} } @article{Sassanfar1993, author = {Sassanfar, M. and Szostak, J. W.}, title = {An RNA motif that binds ATP}, journal = {Nature}, volume = {364}, number = {6437}, pages = {550-3}, note = {Sassanfar, M Szostak, J W eng Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. ENGLAND 1993/08/05 Nature. 1993 Aug 5;364(6437):550-3.}, abstract = {RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.}, keywords = {Adenosine Triphosphate/*metabolism Base Sequence Binding Sites Chromatography Consensus Sequence Dna Molecular Sequence Data Nucleic Acid Conformation RNA/chemistry/isolation & purification/*metabolism NASA Discipline Exobiology NASA Discipline Number 52-20 NASA Program Exobiology Non-NASA Center}, ISSN = {0028-0836 (Print) 0028-0836 (Linking)}, DOI = {10.1038/364550a0}, url = {http://www.ncbi.nlm.nih.gov/pubmed/7687750}, year = {1993}, type = {Journal Article} } @article{Schmidt2000, author = {Schmidt, C. and Welz, R. and Muller, S.}, title = {RNA double cleavage by a hairpin-derived twin ribozyme}, journal = {Nucleic Acids Res}, volume = {28}, number = {4}, pages = {886-94}, note = {Schmidt, C Welz, R Muller, S eng Research Support, Non-U.S. Gov't ENGLAND 2000/01/29 Nucleic Acids Res. 2000 Feb 15;28(4):886-94.}, abstract = {The hairpin ribozyme is a small catalytic RNA that catalyses reversible sequence-specific RNA hydrolysis in trans. It consists of two domains, which interact with each other by docking in an antiparallel fashion. There is a region between the two domains acting as a flexible hinge for interdomain interactions to occur. Hairpin ribozymes with reverse-joined domains have been constructed by dissecting the domains at the hinge and rejoining them in reverse order. We have used both the conventional and reverse-joined hairpin ribozymes for the design of a hairpin-derived twin ribozyme. We show that this twin ribozyme cleaves a suitable RNA substrate at two specific sites while maintaining the target specificity of the individual monoribozymes. For characterisation of the studied ribozymes we have evaluated a quantitative assay of sequence-specific ribozyme activity using fluorescently labelled RNA substrates in conjunction with an automated DNA sequencer. This assay was found to be applicable with hairpin and hairpin-derived ribozymes. The results demonstrate the potential of hairpin ribozymes for multi-target strategies of RNA cleavage and suggest the possibility for employing hairpin-derived twin ribozymes as powerful tools for RNA manipulation in vitro and in vivo.}, keywords = {Base Sequence Hydrolysis Kinetics Molecular Sequence Data Nucleic Acid Conformation RNA/chemistry/*metabolism RNA, Catalytic/*metabolism}, ISSN = {1362-4962 (Electronic) 0305-1048 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/10648779}, year = {2000}, type = {Journal Article} } @article{Schneider2012, author = {Schneider, Gregory F. and Dekker, Cees}, title = {DNA sequencing with nanopores}, journal = {Nat Biotech}, volume = {30}, number = {4}, pages = {326-328}, ISSN = {1087-0156}, DOI = {10.1038/nbt.2181}, url = {http://dx.doi.org/10.1038/nbt.2181}, year = {2012}, type = {Journal Article} } @article{Seeling1999, author = {Seelig, B. and Jaschke, A.}, title = {A small catalytic RNA motif with Diels-Alderase activity}, journal = {Chem Biol}, volume = {6}, number = {3}, pages = {167-76}, note = {Seelig, B Jaschke, A eng Research Support, Non-U.S. Gov't ENGLAND 1999/03/13 Chem Biol. 1999 Mar;6(3):167-76.}, abstract = {BACKGROUND: The 'RNA world' hypothesis requires that RNA be able to catalyze a wide variety of chemical reactions. In vitro selection from combinatorial RNA libraries has been used to identify several catalytic activities, most of which have resulted in a self-modification of RNA at one of its constituents. The formation of carbon-carbon bonds is considered an essential prerequisite for a complex metabolism based on RNA. RESULTS: We describe the selection and characterization of new ribozymes that catalyze carbon-carbon bond formation by Diels-Alder reaction of a biotinylated maleimide with an RNA-tethered anthracene. Secondary structure analysis identified a 49-nucleotide RNA motif that accelerates the reaction about 20,000-fold. The motif has only 11 conserved nucleotides that are present in most of the selected sequences. The ribozyme motif is remarkably adaptable with respect to cofactor and metal-ion requirements. The motif was also re-engineered to give a 38-mer RNA that can act as a 'true' catalyst on short external substrate oligonucleotide-anthracene conjugates. CONCLUSIONS: We have identified a small, highly abundant RNA motif that can solve the complex task of forming two carbon-carbon bonds between two reactants in trans, a catalytic capacity useful for creating prebiotically relevant molecules. This is the smallest and fastest RNA catalyst for carbon-carbon bond formation reported to date.}, keywords = {Algorithms Base Sequence Biotin/metabolism Carbon/metabolism Cloning, Molecular Gene Library Genetic Engineering Kinetics Maleimides/metabolism Molecular Sequence Data Nucleic Acid Conformation RNA, Catalytic/chemistry/genetics/*metabolism}, ISSN = {1074-5521 (Print) 1074-5521 (Linking)}, DOI = {10.1016/S1074-5521(99)89008-5}, url = {http://www.ncbi.nlm.nih.gov/pubmed/10074465}, year = {1999}, type = {Journal Article} } @article{Servanov2007, author = {Serganov, A. and Patel, D. J.}, title = {Ribozymes, riboswitches and beyond: regulation of gene expression without proteins}, journal = {Nat Rev Genet}, volume = {8}, number = {10}, pages = {776-90}, note = {Serganov, Alexander Patel, Dinshaw J eng GM073618/GM/NIGMS NIH HHS/ Comparative Study Research Support, N.I.H., Extramural Review England 2007/09/12 09:00 Nat Rev Genet. 2007 Oct;8(10):776-90. Epub 2007 Sep 11.}, abstract = {Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and external changes. Structural studies have shown how these RNAs can carry out a range of functions. In addition, the contribution of ribozymes and riboswitches to gene expression is being revealed as far more widespread than was previously appreciated. These findings have implications for understanding how cellular functions might have evolved from RNA-based origins.}, keywords = {Animals Binding Sites Catalysis Catalytic Domain Evolution, Molecular *Gene Expression Regulation Humans Models, Molecular Nucleic Acid Conformation RNA/chemistry/metabolism RNA, Catalytic/chemistry/*metabolism *Regulatory Sequences, Ribonucleic Acid Structure-Activity Relationship}, ISSN = {1471-0064 (Electronic) 1471-0056 (Linking)}, DOI = {10.1038/nrg2172}, url = {http://www.ncbi.nlm.nih.gov/pubmed/17846637}, year = {2007}, type = {Journal Article} } @article{Sharmeen1988, author = {Sharmeen, L. and Kuo, M. Y. and Dinter-Gottlieb, G. and Taylor, J.}, title = {Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage}, journal = {J Virol}, volume = {62}, number = {8}, pages = {2674-9}, note = {Sharmeen, L Kuo, M Y Dinter-Gottlieb, G Taylor, J eng CA-06927/CA/NCI NIH HHS/ CC-22651/CC/ODCDC CDC HHS/ RR-05539/RR/NCRR NIH HHS/ Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. 1988/08/01 J Virol. 1988 Aug;62(8):2674-9.}, abstract = {The structure and replication of the single-stranded circular RNA genome of hepatitis delta virus (HDV) are unique relative to those of known animal viruses, and yet there are real similarities between HDV and certain infectious RNAs of plants. Therefore, since some of the latter RNAs have been shown to undergo in vitro site-specific cleavage and even ligation, we tested the hypothesis that similar events might also occur for HDV RNA. In partial confirmation of this hypothesis, we found that in vitro the RNA complementary to the HDV genome, the antigenomic RNA, could undergo a self-cleavage that was not only more than 90% efficient but also occurred only at a single location. This cleavage was found to produce junction fragments consistent with a 5'-hydroxyl and a cyclic 2',3'-monophosphate. Since the observed cleavage was both site-specific and occurred only once per genome length, we propose that the site may be relevant to the normal intracellular replication of the HDV genome. Because the site is located almost adjacent to the 3' end of the delta antigen-coding region, the only known functional open reading frame of HDV, we suggest that the cleavage may have a role not only in genome replication but also in RNA processing, helping to produce a functional mRNA for the translation of delta antigen.}, keywords = {Antigens, Viral/genetics Base Sequence Genes, Viral Hepatitis Delta Virus/*genetics/immunology Molecular Sequence Data Molecular Weight RNA/*metabolism *RNA Processing, Post-Transcriptional RNA, Antisense RNA, Viral/*metabolism}, ISSN = {0022-538X (Print) 0022-538X (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/2455816}, year = {1988}, type = {Journal Article} } @article{Silverman2005, author = {Silverman, S. K.}, title = {In vitro selection, characterization, and application of deoxyribozymes that cleave RNA}, journal = {Nucleic Acids Res}, volume = {33}, number = {19}, pages = {6151-63}, note = {Silverman, Scott K eng Historical Article Research Support, Non-U.S. Gov't Review England 2005/11/16 09:00 Nucleic Acids Res. 2005 Nov 11;33(19):6151-63. Print 2005.}, abstract = {Over the last decade, many catalytically active DNA molecules (deoxyribozymes; DNA enzymes) have been identified by in vitro selection from random-sequence DNA pools. This article focuses on deoxyribozymes that cleave RNA substrates. The first DNA enzyme was reported in 1994 and cleaves an RNA linkage. Since that time, many other RNA-cleaving deoxyribozymes have been identified. Most but not all of these deoxyribozymes require a divalent metal ion cofactor such as Mg2+ to catalyze attack by a specific RNA 2'-hydroxyl group on the adjacent phosphodiester linkage, forming a 2',3'-cyclic phosphate and a 5'-hydroxyl group. Several deoxyribozymes that cleave RNA have utility for in vitro RNA biochemistry. Some DNA enzymes have been applied in vivo to degrade mRNAs, and others have been engineered into sensors. The practical impact of RNA-cleaving deoxyribozymes should continue to increase as additional applications are developed.}, keywords = {Biochemistry/history Biosensing Techniques Catalysis DNA, Catalytic/*chemistry/*metabolism History, 20th Century RNA/chemistry/*metabolism RNA, Messenger/metabolism}, ISSN = {1362-4962 (Electronic) 0305-1048 (Linking)}, DOI = {10.1093/nar/gki930}, url = {http://www.ncbi.nlm.nih.gov/pubmed/16286368}, year = {2005}, type = {Journal Article} } @article{Soukup1999, author = {Soukup, G. A. and Breaker, R. R.}, title = {Nucleic acid molecular switches}, journal = {Trends Biotechnol}, volume = {17}, number = {12}, pages = {469-76}, note = {Soukup, G A Breaker, R R eng Review ENGLAND 1999/11/11 Trends Biotechnol. 1999 Dec;17(12):469-76.}, abstract = {Natural and artificial ribozymes can catalyse a diverse range of chemical reactions. Through recent efforts in enzyme engineering, it has become possible to tailor the activity of ribozymes to respond allosterically to specific effector compounds. These allosteric ribozymes function as effector-dependent molecular switches that could find application as novel genetic-control elements, biosensor components or precision switches for use in nanotechnology.}, keywords = {Allosteric Regulation Base Sequence *Biosensing Techniques DNA/*chemistry Genetic Engineering/*methods/trends Models, Genetic Models, Molecular Molecular Sequence Data Nucleic Acid Conformation Oligonucleotides, Antisense/chemistry/genetics RNA/*chemistry RNA, Catalytic/*chemistry}, ISSN = {0167-7799 (Print) 0167-7799 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/10557159}, year = {1999}, type = {Journal Article} } @article{Sullenger1994, author = {Sullenger, B. A. and Cech, T. R.}, title = {Ribozyme-mediated repair of defective mRNA by targeted, trans-splicing}, journal = {Nature}, volume = {371}, number = {6498}, pages = {619-22}, note = {Sullenger, B A Cech, T R eng Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. ENGLAND 1994/10/13 Nature. 1994 Oct 13;371(6498):619-22.}, abstract = {Ribozymes can be targeted to cleave specific RNAs, which has led to much interest in their potential as gene inhibitors. Such trans-cleaving ribozymes join a growing list of agents that stop the flow of genetic information. Here we describe a different application of ribozymes for which they may be uniquely suited. By targeted trans-splicing, a ribozyme can replace a defective portion of RNA with a functional sequence. The self-splicing intron from Tetrahymena thermophila was previously shown to mediate trans-splicing of oligonucleotides in vitro. As a model system for messenger RNA repair, this group I intron was re-engineered to regenerate the proper coding capacity of short, truncated lacZ transcripts. Trans-splicing was efficient in vitro and proceeded in Escherichia coli to generate translatable lacZ messages. Targeted trans-splicing represents a general means of altering the sequence of specified transcripts and may provide a new approach to the treatment of many genetic diseases.}, keywords = {Animals Base Sequence DNA, Protozoan Escherichia coli/genetics Introns Lac Operon Molecular Sequence Data *RNA Splicing RNA, Catalytic/*metabolism RNA, Messenger/*metabolism RNA, Protozoan Tetrahymena thermophila/genetics/metabolism}, ISSN = {0028-0836 (Print) 0028-0836 (Linking)}, DOI = {10.1038/371619a0}, url = {http://www.ncbi.nlm.nih.gov/pubmed/7935797}, year = {1994}, type = {Journal Article} } @article{Tang1997, author = {Tang, J. and Breaker, R. R.}, title = {Rational design of allosteric ribozymes}, journal = {Chem Biol}, volume = {4}, number = {6}, pages = {453-9}, note = {Tang, J Breaker, R R eng Research Support, Non-U.S. Gov't ENGLAND 1997/06/01 Chem Biol. 1997 Jun;4(6):453-9.}, abstract = {BACKGROUND: Efficient operation of cellular processes relies on the strict control that each cell exerts over its metabolic pathways. Some protein enzymes are subject to allosteric regulation, in which binding sites located apart from the enzyme's active site can specifically recognize effector molecules and alter the catalytic rate of the enzyme via conformational changes. Although RNA also performs chemical reactions, no ribozymes are known to operate as true allosteric enzymes in biological systems. It has recently been established that small-molecule receptors can readily be made of RNA, as demonstrated by the in vitro selection of various RNA aptamers that can specifically bind corresponding ligand molecules. We set out to examine whether the catalytic activity of an existing ribozyme could be brought under the control of an effector molecule by designing conjoined aptamer-ribozyme complexes. RESULTS: By joining an ATP-binding RNA to a self-cleaving ribozyme, we have created the first example of an allosteric ribozyme that has a catalytic rate that can be controlled by ATP. A 180-fold reduction in rate is observed upon addition of either adenosine or ATP, but no inhibition is detected in the presence of dATP or other nucleoside triphosphates. Mutations in the aptamer domain that are expected to eliminate ATP binding or that increase the distance between aptamer and ribozyme domains result in a loss of ATP-specific allosteric control. Using a similar design approach, allosteric hammerhead ribozymes that are activated in the presence of ATP were created and another ribozyme that can be controlled by theophylline was created. CONCLUSIONS: The catalytic features of these conjoined aptamer-ribozyme constructs demonstrate that catalytic RNAs can also be subject to allosteric regulation-a key feature of certain protein enzymes. Moreover, by using simple rational design strategies, it is now possible to engineer new catalytic polynucleotides which have rates that can be tightly and specifically controlled by small effector molecules.}, keywords = {Adenosine/pharmacology Adenosine Triphosphate/metabolism/*pharmacology Allosteric Regulation Base Sequence DNA Primers Electrophoresis, Polyacrylamide Gel Enzyme Inhibitors/pharmacology *Genetic Engineering Kinetics Molecular Sequence Data Mutation Nucleotides/metabolism/pharmacology Polymerase Chain Reaction RNA/genetics/metabolism RNA, Catalytic/chemistry/genetics/*metabolism Theophylline/pharmacology}, ISSN = {1074-5521 (Print) 1074-5521 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/9224568}, year = {1997}, type = {Journal Article} } @article{Travascio1998, author = {Travascio, P. and Li, Y. and Sen, D.}, title = {DNA-enhanced peroxidase activity of a DNA-aptamer-hemin complex}, journal = {Chem Biol}, volume = {5}, number = {9}, pages = {505-17}, note = {Travascio, P Li, Y Sen, D eng Research Support, Non-U.S. Gov't ENGLAND 1998/09/30 Chem Biol. 1998 Sep;5(9):505-17.}, abstract = {BACKGROUND: In vitro selection (SELEX) previously identified short single-stranded DNAs that specifically bound N-methylmesoporphyrin IX (NMM), a stable transition-state analogue for porphyrin-metallation reactions. Interestingly, iron(III)-protoporphyrin (hemin) was a good competitive inhibitor for the DNA-catalyzed metallation reaction, and appeared to bind strongly to the NMM-binding DNA aptamers. We investigated the peroxidase activity of the aptamer-hemin complexes to see if the DNA component of the complex, like the apoenzymes in protein peroxidases, could enhance the low intrinsic peroxidatic activity of hemin. RESULTS: Two porphyrin-binding DNA aptamers bound hemin with submicromolar affinity. The aptamer-hemin complexes had significantly higher peroxidase activity than hemin alone, under physiological conditions. The Vobs of the PS2.M-hemin complex was 250 times greater than that of hemin alone, and significantly superior to a previously reported hemin-catalytic-antibody complex. Preliminary spectroscopic evidence suggests the coordination of the hemin iron in the complex changes, such that the complex more closely resembles horseradish peroxidase and other heme proteins rather than hemin. CONCLUSIONS: A new class of catalytic activity for nucleic acids is reported. The aptamer-hemin complexes described are novel DNA enzymes and their study will help elucidate the structural and functional requirements of peroxidase enzymes in general and the ways that a nucleic acid 'apoenzyme' might work to enhance the intrinsic peroxidatic ability of hemin. These aptamer-hemin complexes could be regarded as prototypes for redox-catalyzing ribozymes in a primordial 'RNA world'.}, keywords = {Catalysis DNA, Single-Stranded/*metabolism Detergents Hemin/*metabolism Hydrogen-Ion Concentration Macromolecular Substances Mesoporphyrins/metabolism Nucleic Acid Conformation Octoxynol Oligonucleotides/*metabolism Peroxidases/*metabolism Spectrophotometry, Atomic Spectrophotometry, Ultraviolet}, ISSN = {1074-5521 (Print) 1074-5521 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/9751647}, year = {1998}, type = {Journal Article} } @article{Tseng2011 author = {Tseng, C. Y. and Ashrafuzzaman, M. and Mane, J. Y. and Kapty, J. and Mercer, J. R. and Tuszynski, J. A.}, title = {Entropic fragment-based approach to aptamer design}, journal = {Chem Biol Drug Des}, volume = {78}, number = {1}, pages = {1-13}, note = {Tseng, Chih-Yuan Ashrafuzzaman, Md Mane, Jonathan Y Kapty, Janice Mercer, John R Tuszynski, Jack A eng Research Support, Non-U.S. Gov't England 2011/04/19 06:00 Chem Biol Drug Des. 2011 Jul;78(1):1-13. doi: 10.1111/j.1747-0285.2011.01125.x. Epub 2011 May 25.}, abstract = {Aptamers are short RNA/DNA sequences that are identified through the process of systematic evolution of ligands by exponential enrichment and that bind to diverse biomolecular targets. Aptamers have strong and specific binding through molecular recognition and are promising tools in studying molecular biology. They are recognized as having potential therapeutic and diagnostic clinical applications. The success of the systematic evolution of ligands by exponential enrichment process requires that the RNA/DNA pools used in the process have a sufficient level of sequence diversity and structural complexity. While the systematic evolution of ligands by exponential enrichment technology is well developed, it remains a challenge in the efficient identification of correct aptamers. In this article, we propose a novel information-driven approach to a theoretical design of aptamer templates based solely on the knowledge regarding the biomolecular target structures. We have investigated both theoretically and experimentally the applicability of the proposed approach by considering two specific targets: the serum protein thrombin and the cell membrane phospholipid phosphatidylserine. Both of these case studies support our method and indicate a promising advancement in theoretical aptamer design. In unfavorable cases where the designed sequences show weak binding affinity, these template sequences can be still modified to enhance their affinities without going through the systematic evolution of ligands by exponential enrichment process.}, keywords = {Aptamers, Nucleotide/*chemistry/pharmacology *Drug Design *Entropy Molecular Dynamics Simulation Phosphatidylserines/chemistry Structure-Activity Relationship Thrombin/chemistry}, ISSN = {1747-0285 (Electronic) 1747-0277 (Linking)}, DOI = {10.1111/j.1747-0285.2011.01125.x}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21496214}, year = {2011}, type = {Journal Article} } @article{Tuerk1990, author = {Tuerk, C. and Gold, L.}, title = {Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase}, journal = {Science}, volume = {249}, number = {4968}, pages = {505-10}, note = {Tuerk, C Gold, L eng GM 19963/GM/NIGMS NIH HHS/ GM 28685/GM/NIGMS NIH HHS/ Research Support, U.S. Gov't, P.H.S. 1990/08/03 Science. 1990 Aug 3;249(4968):505-10.}, abstract = {High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.}, keywords = {Base Sequence *Biological Evolution DNA-Directed DNA Polymerase/*metabolism Escherichia coli/*enzymology *Genes, Viral Genetic Techniques Ligands *Models, Genetic Molecular Sequence Data Nucleic Acid Conformation Polymerase Chain Reaction RNA, Messenger/genetics/*metabolism RNA, Viral/genetics/*metabolism T-Phages/*enzymology Transcription, Genetic}, ISSN = {0036-8075 (Print) 0036-8075 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/2200121}, year = {1990}, type = {Journal Article} } @article{Wang2002, author = {Wang, D. Y. and Lai, B. H. and Feldman, A. R. and Sen, D.}, title = {A general approach for the use of oligonucleotide effectors to regulate the catalysis of RNA-cleaving ribozymes and DNAzymes}, journal = {Nucleic Acids Res}, volume = {30}, number = {8}, pages = {1735-42}, note = {Wang, Dennis Y Lai, Beatrice H Y Feldman, Anat R Sen, Dipankar eng Research Support, Non-U.S. Gov't England 2002/04/09 10:00 Nucleic Acids Res. 2002 Apr 15;30(8):1735-42.}, abstract = {A general approach is described for controlling the RNA-cleaving activity of nucleic acid enzymes (ribozymes and DNAzymes) via the use of oligonucleotide effectors (regulators). In contrast to the previously developed approaches of allosteric and facilitator-mediated regulation of such enzymes, this approach, called 'expansive' regulation, requires that the regulator bind simultaneously to both enzyme and substrate to form a branched three-way complex. Such three-way enzyme-substrate-regulator complexes are catalytically competent relative to the structurally unstable enzyme-substrate complexes. Using the 8-17 and bipartite DNAzymes and the hammerhead ribozyme as model systems, 20- to 30-fold rate enhancements were achieved in the presence of regulators of engineered variants of the above three enzymes, even under unoptimized conditions. Broadly, using this approach ribozyme and DNAzyme variants that are amenable to regulation by oligonucleotide effectors can be designed even in the absence of any knowledge of the folded structure of the relevant ribozyme or DNAzyme. Expansive regulation therefore represents a new and potentially useful technology for both the regulation of nucleic acid enzymes and the detection of specific RNA transcripts.}, keywords = {Base Sequence Catalysis DNA/metabolism DNA, Catalytic/chemistry/genetics/*metabolism Genetic Engineering/*methods Kinetics Macromolecular Substances Models, Genetic Nucleic Acid Conformation Oligonucleotides/*metabolism RNA/metabolism RNA, Catalytic/chemistry/genetics/*metabolism}, ISSN = {1362-4962 (Electronic) 0305-1048 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/11937626}, year = {2002}, type = {Journal Article} } @article{Winz2006, author = {Winz, M. L. and Samanta, A. and Benzinger, D. and Jaschke, A.}, title = {Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry}, journal = {Nucleic Acids Res}, volume = {40}, number = {10}, pages = {e78}, note = {Winz, Marie-Luise Samanta, Ayan Benzinger, Dirk Jaschke, Andres eng Research Support, Non-U.S. Gov't England 2012/02/22 06:00 Nucleic Acids Res. 2012 May;40(10):e78. doi: 10.1093/nar/gks062. Epub 2012 Feb 16.}, abstract = {The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest--of either synthetic or biological origin--is modified at its 3'-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide-alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2'-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2'-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules.}, keywords = {Adenosine Triphosphate/analogs & derivatives/chemistry Azides/chemistry Carbohydrates/chemistry Catalysis Click Chemistry/*methods Copper/*chemistry Escherichia coli/genetics Fluorescent Dyes Nucleotides/chemistry/metabolism Nucleotidyltransferases/metabolism Polynucleotide Adenylyltransferase/*metabolism RNA/*chemistry/metabolism Yeasts/enzymology}, ISSN = {1362-4962 (Electronic) 0305-1048 (Linking)}, DOI = {10.1093/nar/gks062}, url = {http://www.ncbi.nlm.nih.gov/pubmed/22344697}, year = {2012}, type = {Journal Article} } Copper/*chemistry Escherichia coli/genetics Fluorescent Dyes Nucleotides/chemistry/metabolism Nucleotidyltransferases/metabolism Polynucleotide Adenylyltransferase/*metabolism RNA/*chemistry/metabolism Yeasts/enzymology}, ISSN = {1362-4962 (Electronic) 0305-1048 (Linking)}, DOI = {10.1093/nar/gks062}, url = {http://www.ncbi.nlm.nih.gov/pubmed/22344697}, year = {2012}, type = {Journal Article} }