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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 cells<x-ref>Fernandez-Suarez2008</x-ref><x-ref>Tyagi2009</x-ref>. Such methods hold promise to solve the mechanisms of transcription, translation, localization and the function of non-coding RNA. | 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 cells<x-ref>Fernandez-Suarez2008</x-ref><x-ref>Tyagi2009</x-ref>. 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 <i>in vivo</i> and <i>in vitro</i>. Recently, Paige et al. discovered an RNA that mimics the green fluorescent protein (GFP) so called “Spinach” <x-ref>Paige2011</x-ref><x-ref>Strack2013</x-ref><x-ref>Strack2015</x-ref>. 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 cells<x-ref>Buxbaum2015</x-ref><x-ref>Dean2014</x-ref>. Moreover, this RNA has been used as a tool to monitor RNA synthesis in real-time<x-ref>Höfer2013</x-ref><x-ref>Pothoulakis</x-ref> <i>in vitro</i> or to sense different small molecule levels <i>in vivo</i><x-ref>Kellenberger2015</x-ref><x-ref>Kellenberger2013</x-ref>. A prominent example is the sensing of ci-di-GMP concentrations in live cells. To do so, Kellenberger <i>et al.</i> 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. | 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 <i>in vivo</i> and <i>in vitro</i>. Recently, Paige et al. discovered an RNA that mimics the green fluorescent protein (GFP) so called “Spinach” <x-ref>Paige2011</x-ref><x-ref>Strack2013</x-ref><x-ref>Strack2015</x-ref>. 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 cells<x-ref>Buxbaum2015</x-ref><x-ref>Dean2014</x-ref>. Moreover, this RNA has been used as a tool to monitor RNA synthesis in real-time<x-ref>Höfer2013</x-ref><x-ref>Pothoulakis</x-ref> <i>in vitro</i> or to sense different small molecule levels <i>in vivo</i><x-ref>Kellenberger2015</x-ref><x-ref>Kellenberger2013</x-ref>. A prominent example is the sensing of ci-di-GMP concentrations in live cells. To do so, Kellenberger <i>et al.</i> 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. |
Revision as of 14:43, 18 September 2015
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 cells
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.