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

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Single nucleotide polymorphisms (SNPs) are vital for genetic disorder identification, pathogen drug resistance assessment, and infection transmission prevention.
• Precise genotyping using RNA-based probes requires balancing sequence-specific recognition, RNA structural stability, and SNP-induced functional changes.

Purpose of the Study:

• To develop a rapid, specific, and multiplexed method for identifying target sequences with single-nucleotide resolution.
• To create RNA-based aptamer switches (FARSIGHTs) for efficient genotyping and mutation detection.

Main Methods:

• In silico design of aptamer-based RNA switches (FARSIGHTs).
• Demonstration of FARSIGHT probe activation within 5 minutes, independent of upstream amplification.
• Coupling FARSIGHTs with isothermal amplification for sensitive fluorescence-based detection of mutations.

Main Results:

• FARSIGHTs enable rapid (as little as 5 min), low-leakage, and multiplexed identification of target sequences with single-nucleotide specificity.
• Combined FARSIGHTs and isothermal amplification achieve attomolar detection of single nucleotide mutations with high fluorescence output.
• Successfully distinguished SARS-CoV-2 Omicron from Alpha, Beta, and Gamma variants with 100% accuracy in clinical saliva samples.

Conclusions:

• FARSIGHTs offer a versatile platform for rapid and accurate genotyping of virtually any target sequence.
• The technology is easily reprogrammable for emerging pathogens, with potential applications in point-of-care diagnostics and personalized healthcare.