The ability to observe target molecule recognition to individual probe molecules has the potential to dramatically increase the sensitivity of DNA microarrays and next-generation sequencing technology.  We have developed methodology to measure reversible complementary DNA hybridization of fluorescently-labeled target ssDNA to individually-addressed single-stranded probe DNA using relatively simple single-molecule fluorescence imaging techniques.  Substrates are prepared by covalently immobilizing probe ssDNA to random locations on surfaces.  Probe-target hybridization events are located with ~20 nm precision using super-resolution techniques, allowing probes to be addressed at densities near the optical resolution limit, ~ 1.5 mm<sup>-2</sup>, without the need for molecules to be spaced using engineered DNA origami or nanofabricated structures.  Non-specific adsorption is mitigating by back-filling the surrounding substrate with sulfonate groups to passivate the surface, which reduces nonspecific adsorption events to less than 1% of complementary hybridization events.  With such high specificity, labeled probes are not needed to provide additional readout to exclude nonspecific adsorption events, like FRET or co-localization, which eliminates probe photobleaching and allows long observation times.  Micron-scale lateral drift of samples is corrected by spatially correlating the probe-target hybridization locations themselves, negating the need for fiducial markers and their associated two-color imaging systems.  Using this methodology, we observe probe-target recognition for hours, allowing us to identify probe ssDNA molecules on the surface by repeated target hybridization to their locations.  With arrival times and durations for every recognition event, we are able to determine association and dissociation rates for individual probe molecules, and show that association and dissociation rates averaged over all probes match the rates determined from an aggregate of all hybridization events.  This methodology could be adapted to identify a mixture of different target and immobilized-probe ssDNA sequences simultaneously using their unique kinetic signature, without the need for multiple fluorescent labels and multi-color fluorescence imaging.

Super-resolution single-molecule fluorescence imaging of DNA hybridization

251st National Meeting (2016)

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