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 are often limited by a low signal-to-noise ratio, the extensive optimization required 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 biosensors that can be activated by light and small molecules, 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.

Figure 1. Design and biophysical feasibility of activity-responsive RNA polymerases (ARs) based on proximity-dependent split RNAPs

(a) Schematic depiction of AR design. Split T7 RNAP engineered such that it assembles into a functional RNAP when proteins of interest (POIs) fused to each half interact with one another, resulting in transcription of a user-defined sequence of RNA from a supplied DNA substrate. (b) Vectors designed to test split RNAPs in vivo, including a luciferase reporter vector and expression vectors for each of the two halves of the split RNAP. N-terminal split RNAP (red) and C-terminal split RNAP (green) were fused to anti-parallel leucine zipper peptide fusions, ZA (pink), ZB (blue), or ZBneg (gray). ZA and ZB form a tight interaction with one another; ZBneg has three point mutations compared to ZB that dramatically weaken the interaction. (c) Transcriptional output of split RNAPs with fusion proteins assayed in E. coli using the vectors shown in (b). Cells induced for 2 h with arabinose and then analyzed for luminescence (error bars std. error, n = 4). Fusion of the peptides does not interfere with RNAP assembly. Transcription is enhanced if fused peptides interact.

Figure 2. Evolution of a proximity-dependent split RNAP for protein-protein interaction detection

(a) Vectors for PACE system for proximity dependent RNAPs. (b) Schematic of mechanism of PACE system for proximity dependent RNAPs. Phage carry an evolving N-terminal RNAP fused to ZA, which is given a constant choice of assembling with either a C-terminal RNAP variant fused to the ZB binding partner that produces gIII and allows phage replication, or a C-terminal RNAP variant that is fused to the non-interacting ZBneg partner, which poisons phage production by producing a dominant negative form of gIII (gIIIneg). (c) Schematic of the evolution parameters used during PACE. The RBS strengths controlling the expression of the C-terminal RNAPs, the RBSs controlling the output gIII or gIIIneg, and the copy number of the posAP were carefully tuned during the course of the 29 days of evolution. (d) Mapping the mutations of N-29-1 onto T7 RNAP crystal structures (top panel: initiation complex, PDB 1QLN; bottom panel: elongation complex, PDB 1h38). (e) Transcriptional reporter assay of the two primary N-terminal split RNAP genotypes fused to ZA or the evolved ZA double mutant, interacting with either the C-terminal RNAP alone (pink), the C-terminal RNAP fused to ZB (blue), or the C-terminal RNAP fused to ZBneg (grey) (error bars std. error, n = 4). Note: data plotted on a logarithmic scale to show background.

Figure 3. Small molecule and light-responsive ARs

(a) Schematic of light activated RNAP design using the iLID-nano system fused to the evolved split RNAP. (b) Transcription response of the light-activated RNAP system in E. coli. Cells were transformed with expression vectors for the two halves of the light-inducible RNAP and a reporter vector, and then either kept in the dark or illuminated with blue LED light for 3 h prior to transcriptional analysis (error bars std. error, n = 4). (c) Schematic of small molecule responsive RNAP design using FRB and FKBP fused to the evolved split RNAP. (d) Transcription response of the rapamycin-inducible RNAP system in E. coli. Cells were transformed with expression vectors for the two halves of the small molecule-inducible RNAP and a reporter vector, and then induced with varying concentrations of rapamycin for 3 h prior to transcription analysis (error bars std. error, n = 4).

Figure 4. Multidimensional PPI detection by ARs

(a) Design of a synthetic trimolecular protein interaction network, using N-29-1 fused to ZA and FRB (“FZ-N”), and two different binding partners, C-terminal CGG RNAP fused to ZB or ZBneg (“Z-C_G” pr “Z_neg-C_G), and C-terminal T7 RNAP fused to FKBP (“F-C₇”). In the absence of rapamycin, FZ-N/Z-C_G should be the dominant PPI, driving a CGG-promoter output. In the presence of rapamycin, the FZ-N/F-C₇ PPI should also be present. (b) Vectors used to monitor the two PPIs simultaneously as shown in (a). Vectors designed that simultaneously express FZ-N, Z-C_G, and F-C₇, along with T7 promoter-driven luciferase and CGG-promoter-driven DsRed outputs. (c) Simultaneous monitoring both PPIs in the same cells. E. coli transformed with the expression vectors as shown, induced with either DMSO or 10 μM rapamycin for 5 h, then analyzed for luminescence and DsRed fluorescence (error bars std. dev., n = 4).

Figure 5. ARs can trigger a variety of outputs in mammalian cells

(a) Design of the “rapa-T7” vector for rapamycin induced transgene expression in mammalian systems. (b) Validation of the rapa-T7 vector with a fluorescent RNA aptamer as the output. HEK293T cells were transfected with the rapa-T7-F30-2xdBroccoli vector (pJin141) and induced with 0 or 100 nM rapamycin for 30 min in the presence of 20 μM DHFBI-1T then analyzed by fluorescence microscopy. 100 μm scale bar shown. (c) Validation of the rapa-T7 vector with mRNA as the output. HEK293T cells transfected with the rapa-T7-mRNA(GFP) vector (p6-8) were induced with 0 or 10 nM rapamycin overnight and then analyzed by fluorescence microscopy (100 μm scale bar shown). (d) Validation of the rapa-T7 vector with shRNA as the output. HEK293T cells transfected with a GFP expression vector and a rapa-T7-shRNA(GFP) vector (pJin140) and induced with varying concentrations of rapamycin for 44 h. GFP fluorescence analyzed by flow cytometry (error bars std. error, n = 3).