A guide to nucleic acid detection by single-molecule kinetic fingerprinting

Abstract

Conventional methods for detecting small quantities of nucleic acids require amplification by the polymerase chain reaction (PCR), which necessitates prior purification and introduces copying errors. While amplification-free methods do not have these shortcomings, they are generally orders of magnitude less sensitive and specific than PCR-based methods. In this review, we provide a practical guide to a novel amplification-free method, single-molecule recognition through equilibrium Poisson sampling (SiMREPS), that provides both single-molecule sensitivity and single-base selectivity by monitoring the repetitive interactions of fluorescent probes to immobilized targets. We demonstrate how this kinetic fingerprinting filters out background arising from the inevitable nonspecific binding of probes, yielding virtually zero background signal. As practical applications of this digital detection methodology, we present the quantification of microRNA miR-16 and the detection of the mutation EGFR L858R with an apparent single-base discrimination factor of over 3 million.

Digital-resolution detection of microRNA with single-base selectivity by photonic resonator absorption microscopy

“…Unfortunately, we observed high nanoparticle background (>150) when no miR-375 was present. We hypothesized that the nonspecific background was likely due to direct hybridization between unprotected probe bases and the DNA capture (36). To test this, we added a 5-base DNA blocker (10 nM) to the DNA-AuNP/miR mixture, which was designed to bind the 10-base capture strand (36).…”

“…We hypothesized that the nonspecific background was likely due to direct hybridization between unprotected probe bases and the DNA capture (36). To test this, we added a 5-base DNA blocker (10 nM) to the DNA-AuNP/miR mixture, which was designed to bind the 10-base capture strand (36). With the addition of the DNA blocker, we observed <10 counts of nonspecific background in the no-miR-375 case, measured at 2 h. Furthermore, the addition of the DNA blocker did not compromise the ability to detect low concentrations of miR-375 (SI Appendix, Fig.…”

Ultraspecific and Amplification-Free Quantification of Mutant DNA by Single-Molecule Kinetic Fingerprinting

“…ΔG°FP⋅MUT and ΔG°FP⋅WT are the free energies of hybridization of the MUT fluorescent probe (FP) to the MUT and WT targets, respectively, and were estimated using NUPACK with the following parameters: Nucleic acid type = DNA, DNA energy parameters = SantaLucia, 1998, Number of strand species = 2, Maximum complex size = 2, Temperature = 20 °C, [Na + ] = 0.6 M, 1 µM each strand, Dangle treatment = Some. Additional guidelines for SiMREPS probe and assay design can be found elsewhere 21 .…”

Automatic classification and segmentation of single-molecule fluorescence time traces with deep learning

“…The raw movie data are saved and stored in an uncompressed format that takes ~1–10 GB per movie, or perhaps 10–100 GB per experiment. Emission profiles from single fluorophores are localized within the field of view by various spot-finding methods to identify candidate molecules for analysis 9 – 11 . In the case of two-channel measurements such as most smFRET experiments 9 , the signals from the two fluorophores are spectrally separated using a dichroic mirror and projected onto different zones of the camera’s sensor, or onto different cameras, and fluorescent signals originating from the same molecule must be paired up (or colocalized) using one of several image registration methods 12 – 14 .…”

“…This approach permits the observation of equilibrium biomolecular dynamics that would be inaccessible to ensemble techniques 5 . In addition, kinetic fingerprinting approaches such as single-molecule recognition through equilibrium Poisson sampling (SiMREPS) 4,6 employ kinetic probing to achieve highly specific detection of single unlabeled nucleic acids. SiMREPS in particular has been shown to distinguish between a wild-type DNA sequence and C-to-T point mutation 7 with an apparent discrimination factor several orders of magnitude greater than the theoretical maximum for methods relying on thermodynamic discrimination 8 .…”