Single-molecule spectroscopic methods offer the unique ability to measure kinetics at equilibrium by detecting stochastic binding events on a per-molecule basis. We use single-molecule fluorescence imaging to measure hybridization kinetics between individual oligonucleotides immobilized at an interface and fluorescently-labeled complementary DNA in solution. Near the melting temperature, DNA hybridization is reversible, allowing multiple hybridization events to be observed at individual probe molecules, so that their kinetics can be determined. However, the fluorescent labels needed to detect single biomolecules can perturb their binding behavior, and fluorophore photobleaching, photoblinking, etc. can interfere with kinetics measurements. Since target DNA can only hybridize with unhybridized probe DNA, we can use the <i>intervals</i> between events to gain information about the kinetics without the influence of photobleaching, since a hybridized molecule must dissociate before another can hybridize. Because the hybridization kinetics are highly uniform across all immobilized probe DNA, hybridization duration and interval times can be aggregated across all detected molecules to measure kinetic processes with good statistical sampling. By monitoring distributions of hybridization intervals, the kinetics can be measured without tracking molecules for the duration of their bound state, potentially eliminating the influence of photobleaching in single-molecule measurements. In addition, we have developed a competitive assay that allows us to measure the hybridization kinetics of <i>unlabeled</i> target DNA based on its influence on the hybridization rate of labeled “tracer” DNA competing for the same probe DNA. The distribution of association-time intervals between the tracer and probe DNA is affected by competing hybridization with the unlabeled target DNA, allowing us to determine the kinetics unlabeled target hybridization according to a competitive rate model.

DNA hybridization kinetics through analysis of single-molecule binding-time intervals

255th National Meeting (2018)

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