Programmable Fluorescent Aptamer-Based RNA Switches for Rapid Identification of Point Mutations

ABSTRACT 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 in RNA structure is crucial to precise genotyping using RNA-based probes. Here, we report an in silico-designed aptamer-based RNA switch we term FARSIGHT (for Fast Aptamer-based Reporter for SIngle-nucleotide-specific Genotypying through HybridizaTion) that provides rapid, low-leakage, and multiplexed identification of virtually any target sequence in as little as 5 minutes with single-nucleotide specificity. Coupling FARSIGHTs with nucleic acid sequence-based amplification (NASBA) enables robust detection of single-nucleotide mutations at attomolar concentrations with strong fluorescence output. To evaluate these assays, we deploy them to distinguish the SARS-CoV-2 Omicron variant from other SARS-CoV-2 variants (Alpha, Beta, and Gamma) with 100% accuracy on RNA extracted from clinical saliva samples, as confirmed by reverse transcription quantitative polymerase chain reaction and genomic sequencing. FARSIGHTs can thus be easily reprogrammed for genotyping new pathogens with pandemic potential, with potential uses in point-of-care settings for monitoring of emerging infectious diseases and for personalized healthcare applications.