Single-molecule fluorescence microscopy is a proven technique for measuring the kinetics of biomolecule interactions at interfaces, though these experiments must be designed with the intrinsic rates of specific biomolecule interactions in mind. DNA hybridization is difficult to measure using single-molecule techniques because the association rate constant for complementary strands is low, ~106 M-1s-1, requiring long observation times and high solution concentrations of target strand to gather significant statistics. Long exposure to high concentrations of target ssDNA leads to nonspecific adsorption, which is typically mitigated by complex readout sensitive to specific interactions, such as FRET or colocalization. In this work, we describe a scheme for immobilizing probe ssDNA at a glass interface passivated against nonspecific adsorption with anionic blocking groups, yielding surfaces that are sufficiently selective that complementary binding dominates the population of fluorescently-labeled target DNA on the surface. Interactions between probe and target DNA are highly sequence specific; surface populations of hybridized complementary target DNA are nearly 3 orders of magnitude greater than that of a scrambled sequence, eliminating the need for FRET to identify complementary interactions. The surface density of immobilized probe DNA can be controlled from 108 to 1010 cm-2 by the probe concentration during the immobilization reaction. At low density, individual probe molecules can be resolved so that hybridization and dissociation rates can be measured directly from single-molecule trajectories. This assay has the potential to allow for the identification of oligonucleotides out of a mixture of immobilized ssDNA. At higher site densities the substrates are more efficient at capturing target strands from solution, making them excellent ssDNA sensors with very low detection limits. We have developed super-resolution techniques to quantify probe molecule density, so that high-density surfaces could be used to measure equilibria and kinetic constants of weaker interactions, such as short oligonucleotide hybridization and split-aptamer association.

Quantitative single-molecule imaging of DNA hybridization at capture surfaces with sequence specificity

249th National Meeting (2015)

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