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Kinetics and dynamics of oligonucleotide hybridization

Nature Reviews Chemistry volume 9, pages 305–327 (2025)Cite this article

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Abstract

The hybridization of short nucleic acid strands is a remarkable spontaneous process that is foundational to biotechnology and nanotechnology and plays a crucial role in gene expression, editing and DNA repair. Decades of research into the mechanism of hybridization have resulted in a deep understanding of its thermodynamics, but many questions remain regarding its kinetics and dynamics. Recent advances in experiments and molecular dynamics simulations of nucleic acids are enabling more direct insight into the structural dynamics of hybridization, which can test long-standing assumptions regarding its mechanism. In this Review, we summarize the current state of knowledge of hybridization kinetics, discuss the barriers to a molecular description of hybridization dynamics, and highlight the new approaches that have begun uncovering the dynamics of hybridization and the duplex ensemble. The kinetics and dynamics of hybridization are highly sensitive to the composition of nucleic acids, and we emphasize recent discoveries and open questions on the role of nucleobase sequence and chemical modifications.

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Fig. 1: Overview of oligonucleotide hybridization on a two-state free-energy profile.
Fig. 2: Base-pair motions and terminal fraying in the duplex ensemble.
Fig. 3: Kinetics of oligonucleotide hybridization.
Fig. 4: Multiple mechanisms of DNA hybridization observed in coarse-grained molecular dynamics simulations.
Fig. 5: Direct comparison between T-jump infrared spectroscopy and coarse-grained molecular dynamics simulations using MSM relaxation kinetics.
Fig. 6: Dynamics of DNA hybridization and zippering captured with single-molecule imaging and spectroscopy.
Fig. 7: Disruption of base-pairing cooperativity by an AP site.

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Acknowledgements

We acknowledge financial support from the National Institute of General Medical Sciences of the National Institutes of Health (award no. R01-GM118774) and the National Science Foundation under grant no. CHE-2155027. B.A. acknowledges support from the NSF Graduate Research Fellowship Program. We greatly appreciate discussions and feedback on the manuscript from P. Sanstead, M. Jones, M. Todisco, Y. Lee, N. Lewis, J. Schauss, L. Whaley-Mayda and K. Liebl. We thank S. Granick, T. Ouldridge, M. Todisco, H. Wang and M. Woodside for providing original files used in Figs. 3, 4 and 6.

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  1. Brennan Ashwood

    Present address: Department of Chemistry, Columbia University, New York, NY, USA

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  1. Department of Chemistry, The James Franck Institute, and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA

    Brennan Ashwood & Andrei Tokmakoff

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B.A. researched data for the article. Both authors contributed to the discussion of content, wrote the article then reviewed and/or edited the manuscript prior to submission.

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Glossary

All-atom molecular dynamics (MD) simulation

Numerical integration of Newton’s equations of motions for molecules described in terms of forces calculated at the atomic level. A force field is a specific parameterization of an interaction potential between atoms that are used to calculate the forces.

Base-pairing cooperativity

The preference for forming a base pair at the end of an existing, intact segment of the base pairs relative to forming the first base pair in a region of unpaired bases.

Coarse-grained molecular dynamics (MD) simulation

Molecular dynamics simulations where the number of interacting particles are reduced from all atoms to coarse sites with interactions governed by a particular force field.

Diffusive models of reactions

Framework where reactions are modelled as the diffusion of a particle along a free-energy landscape with a characteristic conformational diffusion coefficient that is set by microscopic friction induced by interactions with solvent and intramolecular degrees of freedom.

Ensemble spectroscopy

Spectroscopic methods whose observables arise from a macroscopic sample of many molecules and thereby report on the ensemble average properties of a chemical species. Time-dependent properties of species and reactions are probed by synchronizing the movement of the ensemble of molecules from the reactant states to product states.

Hybridization dynamics

The pathways of time-dependent molecular configurations that oligonucleotides traverse as they transition between single-strand and duplex forms.

Hybridization kinetics

The rates of exchange between duplex, single-strand, and intermediate states and the characterization of energetic barriers separating them.

Markov-state model

A class of kinetic model that uses MD trajectories to identify long-lived molecular states and describes the equilibrium occupancy and probability fluxes between those states.

oxDNA model

Coarse-grained model of DNA that models each nucleotide with three different interaction sites for hydrogen bonding, base stacking and backbone, and employs non-bonded potentials for non-bonded excluded volume, base stacking, cross stacking and electrostatics, as well as a bonded potential between backbone sites. It is parameterized against experimental mechanical properties, experimental hybridization thermodynamics and hybridization thermodynamics predicted by SantaLucia’s empirical nearest-neighbour model.

Single-molecule spectroscopy

Experimental methods that isolate an observable from one molecule and resolve the time-trajectories of individual molecules within an ensemble.

Three-site-per-nucleotide (3SPN.2) model

Coarse-grained model of DNA that uses three interaction sites to represent the phosphate, deoxyribose and nucleobase of each nucleotide, and employs non-bonded potentials for excluded volume, base stacking, cross stacking, base pairing and electrostatics, as well as bonded potential with bond, angle and dihedral contributions. It is parameterized against experimental structural data, duplex melting thermodynamics and free energies of base stacking.

Trajectory

A time-series of periodic observations of a dynamical system. This could be an extended series of experimental observables or a series of molecular structures from a MD dynamics simulation.

Transition paths

Trajectories from a reactant state that successfully cross a free-energy barrier to a reach a product state.

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Ashwood, B., Tokmakoff, A. Kinetics and dynamics of oligonucleotide hybridization. Nat Rev Chem 9, 305–327 (2025). https://doi.org/10.1038/s41570-025-00704-8

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