Programmable fluorescent aptamer-based RNA switches for rapid identification of point mutations

The ability to detect single nucleotide polymorphisms (SNPs) is critical for identifying genetic disorders, assessing pathogen drug resistance and preventing infection transmission. Achieving a delicate balance across sequence-specific recognition, RNA structural stability and functional efficacy based on SNP-induced changes is crucial for precise genotyping using RNA-based probes. Here we report on in silico-designed aptamer-based RNA switches, referred to as 'fast aptamer-based reporters for single-nucleotide-specific identification and genotyping through hybridization' (FARSIGHTs), that enable rapid, low-leakage and multiplexed identification of virtually any target sequence with single-nucleotide specificity. Activation of the FARSIGHT probe can occur in as little as 5 min, separate from upstream amplification. Coupling FARSIGHTs with isothermal amplification enables the robust detection of single nucleotide mutations at attomolar concentrations through strong fluorescence output. We have demonstrated this by distinguishing the SARS-CoV-2 Omicron variant from Alpha, Beta and Gamma with 100% accuracy in RNA from clinical saliva samples. FARSIGHTs can be easily reprogrammed for genotyping emerging pathogens, with potential uses in point-of-care infectious disease monitoring and personalized healthcare applications.