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 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" or the reverse molecular logic "if Y then not 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 vs non-cognate full-length mRNA input. 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.
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