A Panel of Protease-Responsive RNA Polymerases Respond to Biochemical Signals by Production of Defined RNA Outputs in Live Cells

Abstract

RNA is an attractive biomolecule for biosensing and engineering applications due to its information storage capacity and ability to drive gene expression or knockdown. However, methods to link chemical signals to the production of specific RNAs are lacking. Here, we develop protease-responsive RNA polymerases (PRs) as a strategy to encode multiple specific proteolytic events in defined sequences of RNA in live mammalian cells. This work demonstrates that RNAP-based molecular recording devices can be deployed for multimodal analyses of biochemical activities or to trigger gene circuits using measured signaling events.

Evolution of a split RNA polymerase as a versatile biosensor platform

“…Combined with the ability to generate ARs with orthogonal DNA promoter specificity, these tools open the possibility of performing highly multidimensional interaction network analysis, presaging a new approach to cell analysis using high-throughput sequencing 23,24 . ARs should have advantages in terms of sensitivity due to the signal amplification of the RNA output made possible by PCR.…”

“…PACE was carried out to evolve the split T7 RNAP variants using a modified version of previously described methods 24 . E. coli strain S1030 were transformed by electroporation with combinations of the Positive Accessory Plasmid (posAP), Negative Accessory Plasmid (negAP), and Mutagenesis Plasmid (MP) 38 .…”

“…Mixed selection pressures, indicated by listing multiple posAP/negAP sets for a given timepoint, were utilized as appropriate to enhance the likelihood of successful evolution. 24,30,40 …”

“…Inspired by this concept, we recently unveiled protease-responsive RNAPs (PRs) as a new strategy to respond to protease activities by production of defined RNA outputs 24 . We showed that PRs can encode multidimensional protease activities in defined sequences of RNA in both prokaryotic and mammalian cells.…”

RNA Polymerase Tags To Monitor Multidimensional Protein–Protein Interactions Reveal Pharmacological Engagement of Bcl-2 Proteins

“…Moreover, unlike optical reporters, RNAP-based reporters are not limited by spectral overlap considerations, as unique “barcode” sequences can, in principle, be encoded in orthogonal RNA outputs 45,51 . Indeed, we recently demonstrated the possibility of performing multidimensional biochemical assays in mammalian cells using orthogonal protease-responsive RNAPs 52 . Finally, an RNA output offers control over genomic and transcriptomic processing through the production of mRNA, RNAi, or even gRNA, all of which have important applications in genetic screens and synthetic biology 53-57 .…”