First passage time study of DNA strand displacement

Area of Science:

• Molecular Biology
• Biophysics
• Genomics

Background:

• DNA strand displacement is fundamental to genomic functions and DNA engineering.
• Previous bulk studies obscured strand exchange kinetics due to slow association steps.

Purpose of the Study:

• To characterize the kinetics of unimolecular DNA strand displacement at the single-molecule level.
• To resolve the underlying strand exchange dynamics independent of association steps.

Main Methods:

• Utilized a novel single-molecule fluorescence resonance energy transfer (smFRET) assay called the "fission" assay.
• Measured the full distribution of first passage times for strand displacement events.

Main Results:

• The first passage time distribution showed rapid, nearly monotonic decay for a 14-nucleotide displacement domain.
• Mean displacement times averaged 35 ms, with sequence-dependent variations up to 13-fold.
• Displacement kinetics differed between RNA and DNA invaders (except T to U) and complementary invaders.
• Kinetics were largely insensitive to monovalent salt concentrations (0.25-1 M).
• Inferred single-step displacement times of ~30-300 μs using a 1D random walk model.

Conclusions:

• The fission assay provides a powerful framework for dissecting the kinetics of complex molecular processes.
• Unimolecular strand displacement is faster than previously estimated from bulk measurements.
• Sequence identity significantly impacts DNA strand displacement rates, offering insights for DNA engineering.