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In our project we are interested in small molecules that are difficult to sense using common techniques. Here we will describe an innovative system that uses the Spinach2 fused to a specific aptamer to detect small molecules. In 2013 the Jaffrey Lab developed Spinach2 which shows in comparison to Spinach a better folding efficiency and thermostability. <x-ref>Strack2013</x-ref>. To generate aptamers that specifically bind to a small molecule, we will use our software <b>JAWS</b>. Using this set up we will be able to show that our software is capable to support time consuming methods like SELEX, to identify Aptamers that bind specifically to small molecules. As an interesting target, we will sense the small molecule adenosine triphosphate (ATP) in biochemical reactions. A common method that is performed in thousands of laboratories is a <i>in vitro</i> transcriptions To study the function of ribonucleic acids, RNA is generally prepared by <i>in vitro</i> transcription<x-ref>Beckert2011</x-ref>. Using bacteriophage DNA dependent RNA polymerases (T7, T3, Sp6), a variety of different RNAs can be enzymatically synthesized in the lab. In this context we want to establish a new biochemical readout method, called <b>real-time SMS</b>, to record simultaneously small molecules (ATP) and enzymatic kinetics (RNA polymerase) using Spinach2-ATP-Aptamer system.  
 
In our project we are interested in small molecules that are difficult to sense using common techniques. Here we will describe an innovative system that uses the Spinach2 fused to a specific aptamer to detect small molecules. In 2013 the Jaffrey Lab developed Spinach2 which shows in comparison to Spinach a better folding efficiency and thermostability. <x-ref>Strack2013</x-ref>. To generate aptamers that specifically bind to a small molecule, we will use our software <b>JAWS</b>. Using this set up we will be able to show that our software is capable to support time consuming methods like SELEX, to identify Aptamers that bind specifically to small molecules. As an interesting target, we will sense the small molecule adenosine triphosphate (ATP) in biochemical reactions. A common method that is performed in thousands of laboratories is a <i>in vitro</i> transcriptions To study the function of ribonucleic acids, RNA is generally prepared by <i>in vitro</i> transcription<x-ref>Beckert2011</x-ref>. Using bacteriophage DNA dependent RNA polymerases (T7, T3, Sp6), a variety of different RNAs can be enzymatically synthesized in the lab. In this context we want to establish a new biochemical readout method, called <b>real-time SMS</b>, to record simultaneously small molecules (ATP) and enzymatic kinetics (RNA polymerase) using Spinach2-ATP-Aptamer system.  
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<h3 class="basicheader"> Introduction </h3>
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Common techniques to sense small molecules in biochemical reactions are most likely connected to radioactive labeling. The exposure to radioactive sources is known to result in damage of the genetic information and is therefore more and more banished from lab work. However because of its sensitivity, radioactive chemicals are needed to monitor small changes of those molecules.
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A common method that is performed in many laboratories is an <i>in vitro</i> transcription (Fig. 1A). This method is usually performed in a black-box manner. To analyze the decrease of nucleotide trisphosphate over time as well as to determine the success of the <i>in vitro</i> transcription, scientists use radioactive labelled nucleotide triphosphates and perform time demanding acrylamide gel electrophoresis.
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To establish such tool box, two different fluorescent RNA constructs will be applied: The first construct is a fusion of an ATP aptamer<x-ref>Sassanfar1993</x-ref> and Spinach2<x-ref>Strack2013</x-ref> (Fig. 1B), which we will call <b>Spinach2-ATP-Aptamer system </b>. To improve the binding of the ATP aptamer to ATP we apply our own implemented JAWS software. Using our software, nucleotides which form the stem region of the ATP aptamer can be predicted, which will improve binding properties of this RNA to ATP. The fusion of this aptamer to the Spinach2 enables us a fluorescent read out in real-time. Thus, in presence of this small molecule, the ATP aptamer will form a tight stem loop, which results in a new structural conformation of the Spinach aptamer. Finally after binding of the ATP, the Spinach2-ATP-Aptamer is able to interact with the DFHBI. Hereby fluorescence can be measured at 500 nm if the Spinach was excited at 460 nm. We will generate using our <b>JAWS software</b> different Spinach2-ATP-Aptamers. To validate the software as well as to identify the best Spinach2-ATP-Aptamer system, we will analyze them concerning their fluorescent turn-on effect in presence of ATP.
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The second important part of the toolbox is a DNA template containing a promoter according to the applied RNA polymerase and a <b>Malachite Green Aptamer</b> (Fig. 1C), which is excited at 630 nm and emits at 652 nm in presents of malachite green dye. If the Malachite Green Aptamer is transcribed, an increase in fluorescence can be monitored. The second part of the toolbox allows a direct analysis of the success of the <i>in vitro</i> transcription in real-time. In an advanced set up, this system can be extended by DNA template encoding for the RNA of interest (ROI) and a hammerhead ribozyme (HHR). Both fragments are inserted between the promotor and the Malachite Green Aptamer. Inducing the hammerhead ribozyme allows cleavage during the <i>in vitro</i> transcription. Hereby two RNA fragments emerge, the ROI and the hammerhead ribozyme fused to Malachite Green Aptamer (HHR-MGA). Using such setup, the emitted fluorescence of the HHR-MGA will be independent on the applied ROI. Thus the fluorescence induced by the Malachite Green Aptamer can be applied to compare efficiencies of different RNAs at the same time.
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The combination of two aptamers that are able to turn on fluorescence by binding to DFHBI or malachite green provide us the possibility to monitor simultaneously the ATP consumption as well as RNA strand synthesis during <i>in vitro</i> transcription in real-time (Fig. 2). The application of the JAWS software generates us the best ATP-aptamer to identify even small changes during RNA synthesis.
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To validate the JAWS-generated aptamers as well as to show the functionality of the Spinach2-ATP-Aptamer system we will apply different bacteriophage RNA polymerases (T7, Sp6, T3) that are commonly used. In addition inhibitors (Heparin) of polymerases as well as the effect of different buffer compositions will be analyzed by our approach.
 
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Revision as of 11:14, 18 September 2015

Summary

The specific monitoring of small molecules in biochemical reactions is problem that scientist tried to solve since many years. In this project we developed small molecule sensor (SMS) enabling us to analyze biochemical reactions in real-time using a fluorescent readout. Our implemented JAWS software generates us the best ATP-binding Aptamer which is fused to the Spinach Aptamer. In presence of ATP fluorescence will be emitted. This tool enables the sensing of small molecules like ATP that are part of biochemical reactions.

Major Achievements:

  • Validation of the JAWS Software
  • Sensing of ATP concentrations in biochemical reactions
  • Establishment of a dual-fluorescent read-out system to sense small molecules in real-time
  • Detailed monitoring of the ATP consumption as well as RNA strand synthesis

Abstract

The specific monitoring of small molecules in biochemical reactions is problem that scientist tried to solve since many years. In this project we developed small molecule sensor (SMS) enabling us to analyze biochemical reactions in real-time using a fluorescent readout. Using this innovative method, we were able to analyze classical in vitro transcriptions in detail by monitoring the ATP consumption as well as RNA strand synthesis simultaneously. Using a Spinach RNA Aptamer fused to an ATP-binding Aptamer RNA we can specifically sense ATP concentrations in real-time. Our implemented JAWS software generates us the best ATP-binding Aptamer. Thus we can even detect small changes in the concentration of ATP during RNA synthesis. To validate the JAWS software as well as to show the general feasibility of our fluorescent tool-box system we analyzed transcription efficiencies of different RNA polymerases, the influence of the buffer as well as the effect of inhibitors like heparin on transcription. Finally, the combination of the JAWS Software and the fluorescent readout enables the scientific community the possibility to target specifically any small molecule of interest in vivo and in vitro

Introduction

Small molecules are known to regulate many cellular functions. Hence, the development of innovative techniques to analyze metabolic pathways became an important field in research. Those assays require a variety of tools allowing the user to detect small molecules even within live cellsFernandez-Suarez2008Tyagi2009. Such methods hold promise to solve the mechanisms of transcription, translation, localization and the function of non-coding RNA. The most common ways to analyze small molecules or cellular pathways include protein-based methods like GFP or molecule reactive probes which have been engineered in the past. Having a fluorescent readout seems to be a valuable implement to provide time-resolved information in vivo and in vitro. Recently, Paige et al. discovered an RNA that mimics the green fluorescent protein (GFP) so called “Spinach” Paige2011Strack2013Strack2015. This Spinach aptamer was generated by systematic evolution of ligands by exponential enrichment (SELEX). In presence of the 3’5-difluoro-4-hydroxybenzylidne imidazolinone dye (DFHBI), RNA forms a stable Spinach-DFHBI RNA aptamer-complex, which is fluorescent. Since then Spinach has been successfully applied by several laboratories to image RNA in live cellsBuxbaum2015Dean2014. Moreover, this RNA has been used as a tool to monitor RNA synthesis in real-timeHöfer2013Pothoulakis in vitro or to sense different small molecule levels in vivoKellenberger2015Kellenberger2013. A prominent example is the sensing of ci-di-GMP concentrations in live cells. To do so, Kellenberger et al. attached a ci-di-GMP aptamer to the Spinach aptamer. In presence of a small molecule (c-di-GMP) the aptamer forms a functional stem which results in the formation of a fluorescent Spinach-DFHBI RNA aptamer-complex. Thus, small molecule concentrations can be determined by a fluorescence read-out system.

In our project we are interested in small molecules that are difficult to sense using common techniques. Here we will describe an innovative system that uses the Spinach2 fused to a specific aptamer to detect small molecules. In 2013 the Jaffrey Lab developed Spinach2 which shows in comparison to Spinach a better folding efficiency and thermostability. Strack2013. To generate aptamers that specifically bind to a small molecule, we will use our software JAWS. Using this set up we will be able to show that our software is capable to support time consuming methods like SELEX, to identify Aptamers that bind specifically to small molecules. As an interesting target, we will sense the small molecule adenosine triphosphate (ATP) in biochemical reactions. A common method that is performed in thousands of laboratories is a in vitro transcriptions To study the function of ribonucleic acids, RNA is generally prepared by in vitro transcriptionBeckert2011. Using bacteriophage DNA dependent RNA polymerases (T7, T3, Sp6), a variety of different RNAs can be enzymatically synthesized in the lab. In this context we want to establish a new biochemical readout method, called real-time SMS, to record simultaneously small molecules (ATP) and enzymatic kinetics (RNA polymerase) using Spinach2-ATP-Aptamer system.