Abstract
SummaryThe 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.
Highlights
Small genetic variations are major driving forces in biological processes such as evolution and pathogenesis
We have developed a de novodesigned riboregulator termed a single-nucleotide-specific programmable riboregulator (SNIPR) that is capable of differentiating transcript variations down to the single base in living prokaryotic cells and the single functional group in vitro in cell-free systems
Binding of a target RNA with a single-nucleotide change leads to a sequence mismatch and an energy penalty that results in a positive reaction free energy, biasing the riboregulator toward the OFF state
Summary
Small genetic variations are major driving forces in biological processes such as evolution and pathogenesis. Genetic differences down to the single-nucleotide level can have wideranging effects on gene expression, protein folding, and RNA splicing, and lead to far-reaching phenotypic changes, resistance to drugs, and cancer (Syvanen, 2001). Beyond variations at the sequence level, RNA transcripts are subject to an array of chemical modifications that depend on their cellular roles. Molecular probes that recognize single-nucleotide changes and chemical modifications within RNA molecules are valuable tools for understanding cell biology, unearthing cell-to-cell variability, detecting disease, and guiding therapeutic decisions. Such minute changes in sequence and chemistry are very challenging to detect in live cells or for diagnostic purposes when expensive equipment is unavailable
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