Evolution of a split RNA polymerase as a versatile biosensor platform

Abstract

Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies often suffer from a lack of signal-to-noise, requirements for extensive optimization for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting “activity-responsive RNAP” (AR) system to create light and small molecule activated biosensors, demonstrating the “plug-and-play” nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein-protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications.

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

“…We omitted the CGG variant used in the one-by-two interaction analysis because it showed crosstalk with both the CTGA and T3 promoters. Critically, all of the variants, especially those selected for further study, maintained very good dynamic range for PPI detection when paired with the evolved RNAP N and split isoleucine zipper peptides used previously 43,70 , displaying a 134-fold to 300-fold dynamic range (Figure 5C). …”

“…1A). To validate and optimize the protein sensors, we deployed an E. coli -based transcriptional reporter system 43,58 . We cloned expression vectors that constitutively express the BH3 binding domains of tBID and NOXA each fused to the evolved RNAP N tag through a flexible linker.…”

“…The goal would be to allow one BH3-only protein to interact with two different anti-apoptotic proteins in the same cell, such that each interacting pair drives a different RNA output. Previously, we utilized an orthogonal RNAP C 43 , RNAP C (CGG), that selectively transcribes from the “CGG” promoter upon assembly with the RNAP N . In order to deploy this orthogonal system in conjunction with the T7-based system, we cloned a mammalian expression vector that produces Mcl-1-RNAP C (CGG) and RFP expression via the CGG promoter.…”

“…Experiments were conducted as previously described 43 and similar to as noted above. Briefly, S1030 cells 75 were transformed by electroporation with three plasmids: ( i ) a T7 RNAP N -linker-ZA expression plasmid, ( ii ) a ZB-linker-RNAP C expression plasmid with the listed mutations, and ( iii ) a reporter plasmid that encodes luciferase under control of a given target promoter sequence.…”

“…Recently, we developed a proximity-dependent split RNA polymerase (RNAP) reporter system as a new method to measure target interactions in biological systems 43 . This involved evolving an N-terminal split RNAP tag, RNAP N , to assemble with a C-terminal split RNAP tag, RNAP C , in a PPI-dependent manner.…”