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Published December 21, 2018 | Accepted Version + Submitted + Supplemental Material
Journal Article Open

Signal Transduction in Human Cell Lysate via Dynamic RNA Nanotechnology


Dynamic RNA nanotechnology with small conditional RNAs (scRNAs) offers a promising conceptual approach to introducing synthetic regulatory links into endogenous biological circuits. Here, we use human cell lysate containing functional Dicer and RNases as a testbed for engineering scRNAs for conditional RNA interference (RNAi). scRNAs perform signal transduction via conditional shape change: detection of a subsequence of mRNA input X triggers formation of a Dicer substrate that is processed to yield small interfering RNA (siRNA) output anti-Y targeting independent mRNA Y for destruction. Automated sequence design is performed using the reaction pathway designer within NUPACK to encode this conditional hybridization cascade into the scRNA sequence subject to the sequence constraints imposed by X and Y. Because it is difficult for secondary structure models to predict which subsequences of mRNA input X will be accessible for detection, here we develop the RNAhyb method to experimentally determine accessible windows within the mRNA that are provided to the designer as sequence constraints. We demonstrate the programmability of scRNA regulators by engineering scRNAs for transducing in both directions between two full-length mRNAs X and Y, corresponding to either the forward molecular logic "if X then not Y" (X - Y) or the reverse molecular logic "if Y then not X" (Y - X). In human cell lysate, we observe a strong OFF/ON conditional response with low crosstalk, corresponding to a ≈20-fold increase in production of the siRNA output in response to the cognate versus noncognate full-length mRNA input. 2′OMe-RNA chemical modifications protect signal transduction reactants and intermediates against RNase degradation while enabling Dicer processing of signal transduction products. Because diverse biological pathways interact with RNA, scRNAs that transduce between detection of endogenous RNA inputs and production of biologically active RNA outputs hold great promise as a synthetic regulatory paradigm.

Additional Information

© 2018 American Chemical Society. Received: October 9, 2018. Publication Date (Web): December 7, 2018. We thank K. Sakurai and J.J. Rossi for advice on preparation of human cell lysate with functional Dicer, B.R. Wolfe and N.J. Porubsky for assistance with reaction pathway engineering using NUPACK, and P.D. Carlson and J.B. Lucks for discussions on profiling mRNA accessibility. This work was funded by the National Institutes of Health (5R01CA140759), by the National Science Foundation Molecular Programming Project (NSF-CCF-1317694), by the Gordon and Betty Moore Foundation (GBMF2809), by the Rosen Bioengineering Center at Caltech, by a Professorial Fellowship at Balliol College (University of Oxford), and by the Eastman Visiting Professorship at the University of Oxford. The authors declare the following competing financial interest(s): Patents.

Attached Files

Accepted Version - sb8b00424.pdf

Submitted - 439273.full.pdf

Supplemental Material - sb8b00424_si_001.pdf


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August 19, 2023
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