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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 Forster 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.

Point mutations have emerged as prominent biomarkers for disease diagnosis, particularly in the case of cancer. Discovering single-nucleotide variants (SNVs) is also of great importance for the identification of single-nucleotide polymorphisms within the population. The competing requirements of thermodynamic stability and specificity in conventional nucleic acid hybridization probes make it challenging to achieve highly precise detection of point mutants. Here, we present a fluorescence-based assay for low-abundance mutation detection based on toehold-mediated strand displacement and nuclease-mediated strand digestion that enables highly precise detection of point mutations. We demonstrate that this combined assay provides 50-1000-fold discrimination (mean value: 255) between all possible single-nucleotide mutations and their corresponding wild-type sequence for a model DNA target. Using experiments and kinetic modeling, we investigate probe properties that obtain additive benefits from both strand displacement and nucleolytic digestion, thus providing guidance for the design of enzyme-mediated nucleic acid assays in the future.

The detection and quantification of biomarkers have numerous applications in biological research and medicine. The most widely used methods to detect nucleic acids require amplification via the polymerase chain reaction (PCR). However, errors arising from the imperfect copying fidelity of DNA polymerases, limited specificity of primers, and heat-induced damage reduce the specificity of PCR-based methods, particularly for single-nucleotide variants. Furthermore, not all analytes can be amplified efficiently. While amplification-free methods avoid these pitfalls, the specificity of most such methods is strictly constrained by probe binding thermodynamics, which for example hampers detection of rare somatic mutations. In contrast, single-molecule recognition through equilibrium Poisson sampling (SiMREPS) provides ultraspecific detection with single-molecule and single-nucleotide sensitivity by monitoring the repetitive interactions of a fluorescent probe with surface-immobilized targets. In this review, we discuss SiMREPS in comparison with other analytical approaches, and describe its utility in quantifying a range of nucleic acids and other analytes.

Conventional techniques for detecting rare DNA sequences require many cycles of PCR amplification for high sensitivity and specificity, potentially introducing significant biases and errors. While amplification-free methods exist, they rarely achieve the ability to detect single molecules, and their ability to discriminate between single-nucleotide variants is often dictated by the specificity limits of hybridization thermodynamics. Here we show that a direct detection approach using single-molecule kinetic fingerprinting can surpass the thermodynamic discrimination limit by three orders of magnitude, with a dynamic range of up to five orders of magnitude with optional super-resolution analysis. This approach detects mutations as subtle as the drug resistance-conferring cancer mutation EGFR T790M (a single C→T substitution) with an estimated specificity of 99.99999%, surpassing even the leading PCR-based methods and enabling detection of 1 mutant molecule in a background of at least 1 million wild-type molecules. This level of specificity revealed rare, heat-induced cytosine deamination events that introduce false positives in PCR-based detection, but which can be overcome in our approach through milder thermal denaturation and enzymatic removal of damaged nucleobases.

MicroRNAs (miRNAs) have emerged as promising diagnostic biomarkers. We introduce a kinetic fingerprinting approach called Single Molecule Recognition through Equilibrium Poisson Sampling (SiMREPS) for the amplification-free counting of single unlabeled miRNA molecules, which circumvents thermodynamic limits of specificity and virtually eliminates false positives. We demonstrate high-confidence single-molecule detection of synthetic and endogenous miRNAs in both buffer and minimally treated biological liquids, as well as >500-fold discrimination between single nucleotide polymorphisms.

E Unus pluribum, or "Of One, Many", may be at the root of decoding the RNA sequence-structure-function relationship. RNAs embody the large majority of genes in higher eukaryotes and fold in a sequence-directed fashion into three-dimensional structures that perform functions conserved across all cellular life forms, ranging from regulating to executing gene expression. While it is the most important determinant of the RNA structure, the nucleotide sequence is generally not sufficient to specify a unique set of secondary and tertiary interactions due to the highly frustrated nature of RNA folding. This frustration results in folding heterogeneity, a common phenomenon wherein a chemically homogeneous population of RNA molecules folds into multiple stable structures. Often, these alternative conformations constitute misfolds, lacking the biological activity of the natively folded RNA. Intriguingly, a number of RNAs have recently been described as capable of adopting multiple distinct conformations that all perform, or contribute to, the same function. Characteristically, these conformations interconvert slowly on the experimental timescale, suggesting that they should be regarded as distinct native states. We discuss how rugged folding free energy landscapes give rise to multiple native states in the Tetrahymena Group I intron ribozyme, hairpin ribozyme, sarcin-ricin loop, ribosome, and an in vitro selected aptamer. We further describe the varying degrees to which folding heterogeneity impacts function in these RNAs, and compare and contrast this impact with that of heterogeneities found in protein folding. Embracing that one sequence can give rise to multiple native folds, we hypothesize that this phenomenon imparts adaptive advantages on any functionally evolving RNA quasispecies.