Target-dependent RNA polymerase as universal platform for gene expression control in response to intracellular molecules

Fig. 1. Design and characterization of TdRNAP. a Schematic of TdRNAP design and strategy. Split T7 RNAP assembles into a functional RNAP when the fused VH and VL domains interact with molecular targets. The activated RNAP can transcribe genes of interest (GOI) under the control of T7 promoter, resulting in various outputs and applications. b Plasmids used to test GCN4-dRNAP in 293FT cells. N-terminal and C-terminal split RNAP fragments (T7N and T7C) were fused to VL and VH domains of the anti-GCN4 antibody, respectively. c Fluorescence images of 293FT cells transfected with the GCN4-dRNAP and induced with EGFP or EGFP-GCN4. Scale bar, 100 μm. d Dose-dependent transcriptional activation of GCN4-dRNAP. 293FT cells were transfected with the reporter plasmid encoding firefly luciferase and Renilla luciferase under the control of T7 and constitutive promoters, respectively. Cells were then analyzed for luminescence. e Dependency of the antibody binding affinity on the transcription activity of GCN4-dRNAP (left). CDR-H3 sequence and the reported dissociation constant (Kd) of each VH variant (right). The protein and RNA structures were drawn with PDB data (PDB ID: 1QLN, 2Y0G, 1HLL, and 4PHY)…

Fig. 2. Expanding the molecular repertoire for TdRNAP by antibody substitution. a Antibody substitution of TdRNAP using the anti-FLAG and anti-EGFP antibodies, resulting in FLAG- and EGFP-dRNAPs. b Optimization of the framework region of VL domain by CDR grafting. CDRs for the VL domain of the anti-FLAG antibody were grafted to the trastuzumab framework region (CDR-grafted VL domain). c Dose- and affinity-dependent transcriptional activation of FLAG-dRNAP. d Target-specific and dose-dependent transcriptional activation of EGFP-dRNAP. e Validation of the target specificity of GCN4-, FLAG-, and EGFP-dRNAPs by inducing with their corresponding molecular targets. The protein structures were drawn with PDB data (PDB ID: 1QLN, 2Y0G, and 3ADF) (a). Values represent mean ± s.e. of n = 3 biological replicates (b–e). Statistical analysis by unpaired two-tailed t test (b) and one-way ANOVA with Bonferroni correction (e), *P <0.05, **P < 0.01, n.s., not significant (P >0.05). Each P value is listed in Supplementary Table 5. Source data are provided as a Source Data file.

Fig. 3. Design and characterization of RNA and small-molecule-dependent RNAPs. a Design of RNA and small-molecule-dependent RNAPs using the anti-HCV IRES RNA and anti-fluorescein antibodies. b Fluorescence images of 293FT cells transfected with HCV IRES RNA-dRNAP and induced with mono-cistronic or HCV IRES-containing bi-cistronic constructs. Scale bar, 100 μm. c Comparison of the tdTomato fluorescence intensity between cells induced with mono-cistronic and bi-cistronic constructs. The fluorescence intensity is quantified from the integrated density of tdTomato fluorescence. d Fluorescence images of 293FT cells transfected with Fluorescein-dRNAP and induced with 0, 50, or 100 μg/ml fluorescein. Scale bar, 100 μm. e Dose-dependent transcriptional activation of Fluorescein-dRNAP. 293FT cells were transfected with the reporter plasmid encoding firefly luciferase and Renilla luciferase under the control of T7 and constitutive promoters, respectively. The protein and RNA structures were drawn with PDB data (PDB ID: 1QLN and 5A2Q) (a). Values represent mean ± s.e. of n = 3 biological replicates (c, e). Statistical analysis by unpaired two-tailed t test (c) and one-way ANOVA with Dunnett’s…

Fig. 4. Construction of multilayer genetic circuits with TdRNAPs for signal amplification and orthogonal signal transduction. a Design of a transcriptional amplification system using CGG RNAP as an additional output of GCN4-dRNAP. The system involves a T7 promoter-driven CGG RNAP plasmid and a dual promoter reporter plasmid encoding a luciferase reporter gene under the control of both T7 and CGG promoters. The activated GCN4-dRNAP induces the expression of both CGG RNAP and the reporter gene. The reporter gene expression is then further enhanced by the induced CGG RNAP. b Validation of the enhancement of reporter expression by the amplification system in 293FT cells. c Design of an orthogonal genetic circuit consisting of EGFP-dependent T7 RNAP and GCN4-dependent CGG RNAP. Each TdRNAP independently controls the expression of a luciferase reporter gene under its corresponding promoter. EGFP-dependent T7 RNAP and GCN4-dependent CGG RNAP correspond to the expression of firefly and Renilla luciferases, respectively. d Simultaneous monitoring of target-dependent reporter expressions in the same 293FT cells. RLU, relative light units. The protein structures were drawn with PDB data (PDB ID:…

Fig. 5. TdRNAP enables cell-specific genome editing triggered by the detection of the target gene product. a Application of TdRNAP to control genome editing in a target-dependent manner in human cells. GCN4-dRNAP induces gRNA expression when cells express GCN4-fused EGFP (EGFP-GCN4). The gRNA targets and knocks out the EGFP gene by forming a complex with Cas9. b Establishment of the 293FT cell lines carrying EGFP or EGFP-GCN4 gene in the AAVS1 locus. GCN4-dependent EGFP knockout should be triggered preferentially in the cells expressing EGFP-GCN4 than EGFP. c Flow cytometry analysis of the GCN4-dependent EGFP knockout in the established 293FT cell lines. d, e EGFP-negative cell population measured by flow cytometry in EGFP knockout experiments using T7 promoter for GCN4-dependent gRNA expression (d) and U6 promoter for constitutive gRNA expression (e) along with 200 ng of Cas9 expression plasmid. The protein structures were drawn with PDB data (PDB ID: 1QLN, 2Y0G, and 1P4B) (a). Values represent mean ± s.d. of n = 7 (d) and n = 5 (e) biological replicates. Statistical analysis by unpaired two-tailed t test (d, e), **P<0.01, n.s., not significant (P >0.05). Each P value is listed in…