Tethered multifluorophore motion reveals equilibrium transition kinetics of single DNA double helices

Significance Understanding cellular functions and dysfunctions often begins with quantifying the interactions between the binding partners involved in the processes. Learning about the kinetics of the interactions is of particular importance to understand the dynamics of cellular processes. We created a tethered multifluorophore motion assay using DNA origami that enables over 1-hour-long recordings of the statistical binding and unbinding of single pairs of biomolecules directly in equilibrium. The experimental concept is simple and the data interpretation is very direct, which makes the system easy to use for a wide variety of researchers. Due to the modularity and addressability of the DNA origami-based assay, our system may be readily adapted to study various other molecular interactions. We describe a tethered multifluorophore motion assay based on DNA origami for revealing bimolecular reaction kinetics on the single-molecule level. Molecular binding partners may be placed at user-defined positions and in user-defined stoichiometry; and binding states are read out by tracking the motion of quickly diffusing fluorescent reporter units. Multiple dyes per reporter unit enable singe-particle observation for more than 1 hour. We applied the system to study in equilibrium reversible hybridization and dissociation of complementary DNA single strands as a function of tether length, cation concentration, and sequence. We observed up to hundreds of hybridization and dissociation events per single reactant pair and could produce cumulative statistics with tens of thousands of binding and unbinding events. Because the binding partners per particle do not exchange, we could also detect subtle heterogeneity from molecule to molecule, which enabled separating data reflecting the actual target strand pair binding kinetics from falsifying influences stemming from chemically truncated oligonucleotides. Our data reflected that mainly DNA strand hybridization, but not strand dissociation, is affected by cation concentration, in agreement with previous results from different assays. We studied 8-bp-long DNA duplexes with virtually identical thermodynamic stability, but different sequences, and observed strongly differing hybridization kinetics. Complementary full-atom molecular-dynamics simulations indicated two opposing sequence-dependent phenomena: helical templating in purine-rich single strands and secondary structures. These two effects can increase or decrease, respectively, the fraction of strand collisions leading to successful nucleation events for duplex formation.