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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.

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Figure 1: Design and biophysical feasibility of ARs based on proximity-dependent split RNAPs.
Figure 2: Evolution of a proximity-dependent split RNAP for PPI detection.
Figure 3: Small-molecule- and light-responsive ARs.
Figure 4: Multidimensional PPI detection by ARs.
Figure 5: ARs can trigger a variety of outputs in mammalian cells.

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Acknowledgements

We thank I. Chronis (University of Chicago), Y. Koh (University of Chicago), and D. Ahn (University of Chicago) for technical assistance, and D. Liu (Harvard University), B. McNaughton (Colorado State University), A. Deiters (University of Pittsburgh), B. Glick (University of Chicago), M. Glotzer (University of Chicago), Y. Krishnan (University of Chicago), J. Thornton (University of Chicago), and Y. Weizmann (University of Chicago) for supplying equipment and materials. This work was supported by the University of Chicago, the Cancer Research Foundation, the National Institute of General Medical Sciences of the National Institutes of Health (R35 GM119840) to B.C.D., the University of Chicago Medicine Comprehensive Cancer Center (P30 CA14599), and the National Center for Advancing Translational Sciences of the National Institutes of Health (UL1 TR000430). J.Z.-B. was supported by a Chemical Biology Training Grant from the US National Institutes of Health (T32GM008720).

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J.Z.-B., J.P., and B.C.D. cloned and validated all materials and performed transcription assays. J.P. and B.C.D. performed PACE experiments. J.Z.-B. and J.P. performed cell culture experiments. J.Z.-B., J.P., and B.C.D. designed experimental strategies and wrote the paper.

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Correspondence to Bryan C Dickinson.

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The authors have filed a provisional patent application on the AR system.

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Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–6. (PDF 4992 kb)

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Pu, J., Zinkus-Boltz, J. & Dickinson, B. Evolution of a split RNA polymerase as a versatile biosensor platform. Nat Chem Biol 13, 432–438 (2017). https://doi.org/10.1038/nchembio.2299

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