Ligand mediated transcriptional regulation using DNA aptamers in cell free systems

Abstract

Engineering gene networks offers an opportunity to harness biological function for biotechnological and biomedical applications. Critical to these efforts is the availability of a large library of ligand sensitive gene regulatory systems and the ability to rapidly test and evaluate these networks. When compared to the use of live cells, designing such systems in a cell free context offers a potentially simpler and more flexible context. Here we describe a strategy to regulate T7 RNA polymerase using DNA aptamers in a cell free context. We test the hypothesis that a DNA aptamer, when placed near the transcription start site, interferes with transcription in the presence of the target molecule. We utilized a DNA aptamer that binds thrombin as a model system for demonstrating the feasibility of the approach. We show that for the hybrid T7‐aptamer promoter, thrombin addition results in up to a 5‐fold reduction in gene expression. We further demonstrate that gene expression is dependent on the thrombin concentration and can be tuned by altering the position of the aptamer relative to the transcription start site. These results pave the way for expanding the library of ligands that can be used for regulating gene expression and can enable facile engineering of gene networks in cell free systems. This research was supported by NIH Grant EB000657