Precise and Programmable Detection of Mutations Using Ultraspecific Riboregulators

Summary 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.
Graphical Abstract
Highlights • SNIPRs are riboregulators with single-nucleotide-specific RNA detection capabilities • They operate in live cells and in paper-based cell-free reactions • Automated in silico design allows SNIPRs to detect many different harmful mutations • Isothermal SNIPR tests enable portable human genotyping and viral strain detection
The development of high-specificity RNA toehold-based sensors allows for the discrimination of point mutations and RNA base modifications and enables genotypic diagnosis for E. coli, human samples, and Zika virus strains