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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. | 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. | 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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Revision as of 12:56, 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 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.