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Precise and Programmable Detection of Mutations Using Ultraspecific Riboregulators.

Cell (2020-02-29)
Fan Hong, Duo Ma, Kaiyue Wu, Lida A Mina, Rebecca C Luiten, Yan Liu, Hao Yan, Alexander A Green
ABSTRACT

The ability to identify single-nucleotide mutations is critical for probing cell biology and for precise detection of disease. However, the small differences in hybridization energy provided by single-base changes makes identification of these mutations challenging in living cells and complex reaction environments. Here, we report a class of de novo-designed prokaryotic riboregulators that provide ultraspecific RNA detection capabilities in vivo and in cell-free transcription-translation reactions. These single-nucleotide-specific programmable riboregulators (SNIPRs) provide over 100-fold differences in gene expression in response to target RNAs differing by a single nucleotide in E. coli and resolve single epitranscriptomic marks in vitro. By exploiting the programmable SNIPR design, we implement an automated design algorithm to develop riboregulators for a range of mutations associated with cancer, drug resistance, and genetic disorders. Integrating SNIPRs with portable paper-based cell-free reactions enables convenient isothermal detection of cancer-associated mutations from clinical samples and identification of Zika strains through unambiguous colorimetric reactions.

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Sigma-Aldrich
Chlorophenol Red-β-D-galactopyranoside, ≥90% (HPLC)