@article{wang_highly_2015, title = {Highly sensitive chemiluminescent detection of lead ion based on its displacement of potassium in G-Quadruplex {DNAzyme}}, volume = {140}, issn = {1364-5528}, doi = {10.1039/c5an00884k}, abstract = {A simple and highly sensitive method for detecting lead ion (Pb(2+)) in biosamples was developed based on its displacement of potassium in G-Quadruplex {DNAzyme}, which can catalyze the luminol-H2O2 chemiluminescence ({CL}) reaction. By introducing a G-rich {DNA} sequence, {PS}2.M, which can fold into a G-quadruplex when binding with hemin in the presence of K(+) and act as a superior horseradish peroxidase ({HRP}) mimicking-enzyme, we found this {DNAzyme} can effectively catalyze the H2O2-mediated oxidation of luminol, resulting in strong {CL} emission. The K(+)-stabilized G-quadruplex, upon the addition of Pb(2+), is transformed into a Pb(2+)-stabilized G-quadruplex with higher stability but poor {DNAzyme} activity, sharply decreasing the {CL} readout signal. With this, a simple and sensitive detection method for Pb(2+) in biosamples such as human hairs was developed with a linear range of 0.4-10 {nM} Pb(2+) and a limit of detection (3σ) of 0.06 {nM}. Owing to the introduction of G-quadruplex {DNAzyme}, which was employed not only as a sensing unit but also as a catalyst in the chemiluminescent assay, this method holds great potential for clinical plumbism diagnosis by testing hair.}, pages = {5742--5747}, number = {16}, journaltitle = {The Analyst}, shortjournal = {Analyst}, author = {Wang, Hong and Wang, Dong Mei and Huang, Cheng Zhi}, date = {2015-07-27}, pmid = {26165488} } @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},@articl@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}, }