First Passage Time Study of DNA Strand Displacement

Abstract

DNA strand displacement is a process by which a DNA duplex is invaded by a single-stranded nucleic acid, annealing to one strand and displacing the other. Strand displacement has many applications in DNA nanoengineering and is fundamental to biological processes such as homologous recombination and the repair of double strand breaks. Strand displacement has previously been studied in bulk; however, a slow, bimolecular toehold formation step obscures the kinetics of displacement in bulk studies. Here, we describe a single-molecule FRET assay dubbed “fission” which allows us to study the first passage time of strand displacement directly. We report an average recorded displacement time of 35ms for short (14bp) invasion domains. We show that strand displacement times depend on the sequence of the invader, differing by up to a factor of 13, and that complimentary invaders can yield different mean displacement times. We demonstrate that extending the length of the invading strand slows down invasion. Substituting DNA invaders with RNA, with identical sequence except for a T->U substitution, also affects invasion times. The salt concentration of the buffer, however, does not appear to have an effect. Finally, we fit a one-dimensional random walk model to our data, providing invasion rates for each of the four DNA bases.