Evolution of an inhibitory RNA aptamer against T7 RNA polymerase

Aptamers are promising gene components that can be used for the construction of synthetic gene circuits. In this study, we isolated an RNA aptamer that specifically inhibits transcription of T7 RNA polymerase (RNAP). The 38-nucleotide aptamer, which was a shortened variant of an initial SELEX isolate, showed moderate inhibitory activity. By stepwise doped-SELEX, we isolated evolved variants with strong inhibitory activity. A 29-nucleotide variant of a doped-SELEX isolate showed 50% inhibitory concentration at 11 nM under typical in vitro transcription conditions. Pull-down experiments revealed that the aptamer inhibited the association of T7 RNAP with T7 promoter DNA.

▸ An inhibitory RNA aptamer against T7 RNA polymerase was isolated. ▸ Evolved aptamers with strong inhibitory activity were isolated by doped-SELEX. ▸ Aptamer sequence/structure requirements were elucidated based on phylogeny. ▸ A 29-nucleotide variant of an evolved aptamer has an IC50 of 11 nM. ▸ The aptamer inhibits the association of T7 RNA polymerase with T7 promoter DNA.