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.