(16b) Single Molecule Studies of Ionic Strength Effects on Interfacial DNA Searching and Hybridization

Authors: 
Traeger, J. - Presenter, University of Colorado, Boulder
Schwartz, D. K., University of Colorado Boulder
Hybridization between soluble ssDNA strands (“target”) with surface-immobilized complimentary oligonucleotides (“probe”) is an important function of many biotechnologies, and electrostatic repulsion between negatively-charged strands can inhibit surface hybridization kinetics such that electrostatic screening by salt counterions is required to induce hybridization. Changes in ionic strength in these systems have been shown to induce complex changes in DNA properties that may alter surface kinetics such as strand conformation, strand-surface interactions, and nonspecific interactions between probes and the surface. These complex changes may alter surface adsorption kinetics, molecular searching behavior, and the stability of associated complementary DNA pairs, affecting surface hybridization equilibria.

In this work, we utilize single-molecule total internal fluorescence microscopy to characterize the effect of electrostatic screening on oligonucleotide transport and hybridization at a solid-liquid interface by introducing dissolved DNA to these surfaces at various ionic strengths and a range of surface grafting density of immobilized probes. Through molecular tracking, we are able to characterize heterogeneous searching behaviors of molecules via anomalous surface diffusion. We correlate changes in molecular transport with changes in association kinetics between DNA strands, allowing us to assess how transport limitations affect successful hybridization. This provides insight into the specific mechanisms of surface hybridization that are affected by electrostatic repulsion.