Diversifying Substrates and Reaction Conditions for Polymerase Strand Recycling.

Cell-free biosensing systems are being engineered as versatile and programmable diagnostic technologies. A core component of cell-free biosensors is programmable molecular circuits that improve biosensor speed, sensitivity, and specificity by performing molecular computations such as logic evaluation and signal amplification. In previous work, we developed one such circuit system called Polymerase Strand Recycling (PSR), which amplifies cell-free molecular circuits by using T7 RNA polymerase off-target transcription to recycle nucleic acid inputs. We showed that PSR circuits can be configured to detect RNA target inputs as well as be interfaced with allosteric transcription factor-based biosensors to amplify signals and enhance sensitivity. Here we expand the development of PSR circuit empirical design guidelines to generalize the platform for detecting a diverse set of microRNA inputs. We show that PSR circuit function can be enhanced through engineering T7 RNAP, and we present troubleshooting strategies to optimize PSR circuit performance.