Multifactorial Modulation of Binding and Dissociation Kinetics on Two-Dimensional DNA Nanostructures

We use single-particle fluorescence resonance energy transfer (FRET) to show that organizing oligonucleotide probes into patterned two-dimensional arrays on DNA origami nanopegboards significantly alters the kinetics and thermodynamics of their hybridization with complementary targets in solution. By systematically varying the spacing of probes, we demonstrate that the rate of dissociation of a target is reduced by an order of magnitude in the densest probe arrays. The rate of target binding is reduced less dramatically, but to a greater extent than reported previously for one-dimensional probe arrays. By additionally varying target sequence and buffer composition, we provide evidence for two distinct mechanisms for the markedly slowed dissociation: direct hopping of targets between adjacent sequence-matched probes and nonsequence-specific, salt-bridged, and thus attractive electrostatic interactions with the DNA origami pegboard. This kinetic behavior varies little between individual copies of a given array design and will have significant impact on hybridization measurements and overall performance of DNA nanodevices as well as microarrays.