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Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery

Abstract

Synthetic regulatory circuits encoded in RNA rather than DNA could provide a means to control cell behavior while avoiding potentially harmful genomic integration in therapeutic applications. We create post-transcriptional circuits using RNA-binding proteins, which can be wired in a plug-and-play fashion to create networks of higher complexity. We show that the circuits function in mammalian cells when encoded in modified mRNA or self-replicating RNA.

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Figure 1: RNA-only multi-input microRNA classifier circuit differentiates between HeLa, HEK293 and MCF7 cells.
Figure 2: Post-transcriptional cascades and two-state switch.

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Acknowledgements

We thank K. Hayashi and N. Nishimura (Kyoto University) for supporting modified mRNA experiments. We also thank A.C. Goldstrohm and C. Weidmann (University of Michigan Medical School) for sharing Drosophila constructs containing MS2-fused repressors and the corresponding reporter, X. Zhang (MIT) and O. Andries (Ghent University) for assisting in VEE replicon construction, T.E. Wagner, D. Densmore (Boston University) and S. Payne (MIT) for communicating results before publication, W.M. Gelbart, C.M. Knobler, A. Berk, O. Azizgolshani and J.M. Parker (University of California, Los Angeles) for sharing replicon constructs and expertise, as well as A. Graziano (MIT) for supporting pDNA experiments and H. Chung (Harvard University) for designing the intronic mKate construct. This work was supported by Defense Advanced Research Projects Agency DARPA-BAA-11-23, US National Institutes of Health grants no. P50-GM098792, 5-R01-CA155320-03, National Science Foundation GRFP grant no. 1122374 (R.W.) and JSPS KAKENHI grant numbers 23681042, 24104002, and Research Center Network for Realization of Regenerative Medicine from the Japan Science and Technology Agency (H.S.).

Author information

Authors and Affiliations

Authors

Contributions

L.W. and R.W. conceived the ideas, L.W. designed and performed pDNA experiments, K.E. designed and performed modRNA experiments, T.K. designed and performed replicon experiments, V.S. designed and performed pDNA apoptotic assay and 3′ UTR repressor test in HeLa cells. B.S. created computational model of pDNA and replicon-based switch. L.W., R.W., K.E. and H.S. wrote the manuscript with input from all other authors.

Corresponding authors

Correspondence to Hirohide Saito or Ron Weiss.

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Competing interests

H.S., K.E., R.W., L.W., T.K. and V.S. are co-inventors on patent applications covering the RNA circuits described here.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–28, Supplementary Tables 1–6 and Supplementary Notes 1–3 (PDF 14585 kb)

Supplementary Source Code

RNA circuits model (TXT 22 kb)

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Wroblewska, L., Kitada, T., Endo, K. et al. Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery. Nat Biotechnol 33, 839–841 (2015). https://doi.org/10.1038/nbt.3301

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