A guide to accelerated direct digital counting of single nucleic acid molecules by FRET-based intramolecular kinetic fingerprinting.

Cell-free nucleic acids (cfNAs) such as short non-coding microRNA (miRNA) and circulating tumor DNA (ctDNA) that reside in bodily fluids have emerged as potential cancer biomarkers. Methods for the rapid, highly specific, and sensitive monitoring of cfNAs in biofluids have, therefore, become increasingly attractive as clinical diagnosis tools. As a next generation technology, we provide a practical guide for an amplification-free, single molecule Förster resonance energy transfer (smFRET)-based kinetic fingerprinting approach termed intramolecular single molecule recognition through equilibrium Poisson sampling, or iSiMREPS, for the rapid detection and counting of miRNA and mutant ctDNA with virtually unlimited specificity and single molecule sensitivity. iSiMREPS utilizes a pair of fluorescent detection probes, wherein one probe immobilizes the target molecules on the surface, and the other probe transiently and reversibly binds to the target to generate characteristic time-resolved fingerprints as smFRET signal that are detected in a total internal reflection fluorescence microscope. Analysis of these kinetic fingerprints enables near-perfect discrimination between specific binding to target molecules and nonspecific background binding. By accelerating kinetic fingerprinting using the denaturant formamide and reducing background signals by removing target-less probes from the surface via toehold-mediated strand displacement, iSiMREPS has been demonstrated to count miR-141 and EGFR exon 19 deletion ctDNA molecules with a limit of detection (LOD) of ~1 and 3 fM, respectively, as well as mutant allele fractions as low as 0.0001%, during a standard acquisition time of only ~10 s per field of view. In this review, we provide a detailed roadmap for implementing iSiMREPS more broadly in research and clinical diagnostics, combining rapid analysis, high specificity, and high sensitivity.