A precise understanding of DNA hybridization kinetics is essential for predicting the temperature-dependent dynamics of DNA-based nano systems. While the equilibrium thermodynamics of DNA duplex formation and its strong sequence dependence are comprehensively understood, a detailed experimental characterization and theoretical understanding of the underlying kinetics remain incomplete. To comprehensively evaluate the sequence-dependent hybridization kinetics we here employ temperature-dependent Single-molecule parallel analysis for rapid exploration of sequence space (Thermo-SPARXS) measurements which integrate single-molecule fluorescence microscopy at variable temperatures with high-throughput sequencing. Using this platform, we quantified temperature-dependent association and dissociation rates for an orthogonal library of seventy-nine 10-bp duplexes between 21°C and 40°C in a single experiment. We could resolve changes of the dissociation rates over two orders of magnitude that increased exponentially with increasing temperature, while the association rates increased only slightly. To interpret these results, we solved a kinetic zipper model for DNA hybridization that included all possible base-pairing states. Despite the complexity of the reaction network, the model predicts that association and dissociation kinetics are controlled by a single rate-limiting step, with the dissociation rate scaling exponentially with the base-pairing free energy. Fitting the experimental data with the model provided the essential rate constants for DNA hybridization, revealing that the DNA duplex extension/shortening steps rather than duplex nucleation are rate limiting under our conditions. Altogether, our work provides a quantitative framework to predict the sequence-dependent kinetics of DNA hybridization in the absence of alternative secondary structures.
Kumar et al. (Sun,) studied this question.