First passage time study of DNA strand displacement

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 R-loop formation. Strand displacement has previously been studied in bulk; however, its kinetics is obscured by a slow, bimolecular toehold formation step. Here, we describe a single-molecule FRET assay dubbed “fission” which allows us to study the first passage time of strand displacement directly. The measured displacement time for a 14-bp domain is on average ~30 ms, with as much as ~10-fold variation among different invader sequences tested. In contrast to DNA duplex stability, the measured displacement time is relatively insensitive to monovalent salt concentration. We also show 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. Finally, we fit a one-dimensional random walk model to our data, providing invasion rates for each of the four DNA bases.