sb8b00424_si_001.pdf (2.77 MB)
Signal Transduction in Human Cell Lysate via Dynamic RNA Nanotechnology
journal contribution
posted on 2018-12-06, 20:50 authored by Lisa M. Hochrein, Tianjia J. Ge, Maayan Schwarzkopf, Niles A. PierceDynamic
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.
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Keywords
OFFcell lysatereaction pathway designerNUPACKmRNA input X triggers formationdetectionsequence constraintsDicerengineering scRNAsAutomated sequence designmRNA input Xsignal transduction reactantsHuman Cell LysatescRNA sequence subjectsignal transduction productsXDynamic RNA Nanotechnology Dynamic RNA nanotechnologyY
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