Is DNA Branch Migration a One-Dimensional Random Walk?

Abstract

Toehold-mediated DNA strand displacement is a ubiquitous tool used in various applications of nanotechnology including biosensors, DNA walkers, and DNA computing. More fundamentally, DNA strand displacement is a crucial step in homologous recombination and DNA repair. The reaction involves 3 strands of DNA whereby initially 2 form a partially bound complementary duplex. The third strand, which is longer and entirely complementary, is thought to first bind to the toehold, the unpaired region of partially bound duplex. It has long been thought that the 2 complementary strands compete for base pairs at a junction or “branch” which undergoes a one-dimensional random walk until one of the strands is completely displaced. However, direct experimental proof of this concept has never been demonstrated. In this study, we investigate the kinetics of three-strand branch migration to address the suitability of the random walk model. We used single-molecule FRET and fluorescence dequenching to construct the full distribution of displacement times. We explore the effects of salt, sequence, and length on displacement kinetics to illuminate the underlying mechanism of DNA strand displacement. Our results show that the random walk model cannot adequately describe the observed kinetics of DNA strand displacement and implicate cooperativity in branch migration.