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.