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ACS Synth Biol. 2018 Nov 20. doi: 10.1021/acssynbio.8b00288. [Epub ahead of print]

Nucleic Acid Strand Displacement with Synthetic mRNA Inputs in Living Mammalian Cells.

Author information

1
Department of Bioengineering , University of Washington , Seattle , Washington 98105 , United States.
2
Department of Electrical Engineering , University of Washington , Seattle , Washington 98195 , United States.
3
Paul G. Allen School of Computer Science & Engineering , University of Washington , Seattle , Washington 98195 , United States.

Abstract

Strand displacement reactions are widely used in DNA nanotechnology as a building block for engineering molecular computers and machines. Here, we demonstrate that strand displacement-based probes can be triggered by RNA expressed in mammalian cells, thus taking a step toward adapting the DNA nanotechnology toolbox to a cellular environment. We systematically compare different probe architectures in order to identify a design that works robustly in living cells. Our optimized strand displacement probe combines chemically modified nucleic acids that enhance stability to degradation by cellular nucleases with structural elements that improve probe retention in the cytoplasm. We visualize probe binding to individual mRNA carrying 96 repeats of a target sequence in the 3'UTR. We find that RNA counts based on live cell imaging using a strand displacement probe are comparable to counts from independent measurement based on fluorescence in situ hybridization experiments. We used probes with scrambled toeholds and scrambled binding domains to demonstrate that target recognition indeed occurs through toehold-mediated strand displacement. Our results demonstrate that strand displacement probes can work reliably in mammalian cells and lay the groundwork for future applications of such probes for live-cell imaging and molecular computing.

PMID:
30441897
DOI:
10.1021/acssynbio.8b00288

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