Rapid kinetic fingerprinting of single nucleic acid molecules by a FRET-based dynamic nanosensor

Biofluid-derived cell-free nucleic acids such as microRNAs (miRNAs) and circulating tumor-derived DNAs (ctDNAs) have emerged as promising disease biomarkers. Conventional detection of these biomarkers by digital PCR and next generation sequencing, although highly sensitive, requires time-consuming extraction and amplification steps that also increase the risk of sample loss and cross-contamination. To achieve the direct, rapid, and amplification-free detection of miRNAs and ctDNAs with near-perfect specificity and single-molecule level sensitivity, we herein designed a single-molecule kinetic fingerprinting assay, termed intramolecular single-molecule recognition through equilibrium Poisson sampling (iSiMREPS). iSiMREPS exploits a dynamic DNA nanosensor comprising a surface anchor and a pair of fluorescent detection probes: one probe captures a target molecule onto the surface, while the other transiently interrogates the target to generate kinetic fingerprints by intramolecular single-molecule Förster resonance energy transfer (smFRET) that are recorded by single-molecule fluorescence microscopy and identify the target after kinetic filtering and data analysis. We optimize the sensor design, use formamide to further accelerate the fingerprinting kinetics, and maximize sensitivity by removing non-target-bound probes using toehold-mediated strand displacement to reduce background. We show that iSiMREPS can detect, in as little as 10 s, two distinct, promising cancer biomarkers-miR-141 and a common EGFR exon 19 deletion-reaching a limit of detection (LOD) of ~3 fM and a mutant allele fraction among excess wild-type as low as 1 in 1 million, or 0.0001%. We anticipate that iSiMREPS will find utility in research and clinical diagnostics based on its features of rapid detection, high specificity, sensitivity, and generalizability.