Thrombin-Mediated Transcriptional Regulation Using
DNA Aptamers in DNA-Based Cell-Free Protein Synthesis
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Abstract
Realizing the potential of cell-free
systems will require development
of ligand-sensitive gene promoters that control gene expression in
response to a ligand of interest. Here, we describe an approach to
designing ligand-sensitive transcriptional control in cell-free systems
that is based on the combination of a DNA aptamer that binds thrombin
and the T7 bacteriophage promoter. Placement of the aptamer near the
T7 promoter, and using a primarily single-stranded template, results
in up to a 6-fold change in gene expression in a ligand concentration-dependent
manner. We further demonstrate that the sensitivity to thrombin concentration
and the fold change in expression can be tuned by altering the position
of the aptamer. The results described here pave the way for the use
of DNA aptamers to achieve modular regulation of transcription in
response to a wide variety of ligands in cell-free systems