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1. Evaluating the effect of ionic strength on PNA:DNA duplex formation kinetics

   https://doi.org/10.1039/D1CB00025J  

   Swenson, Colin S.; Lackey, Hershel H.; Reece, Eric J.; Harris, Joel M.; Heemstra, Jennifer M.; Peterson, Eric M. (January 2021, RSC Chemical Biology)

   null (Ed.)

   Peptide nucleic acid (PNA) is a unique synthetic nucleic acid analog that has been adopted for use in many biological applications. These applications rely upon the robust Franklin–Watson–Crick base pairing provided by PNA, particularly at lower ionic strengths. However, our understanding of the relationship between the kinetics of PNA:DNA hybridization and ionic strength is incomplete. Here we measured the kinetics of association and dissociation of PNA with DNA across a range of ionic strengths and temperatures at single-molecule resolution using total internal reflection fluorescence imaging. Unlike DNA:DNA duplexes, PNA:DNA duplexes are more stable at lower ionic strength, and we demonstrate that this is due to a higher association rate. While the dissociation rate of PNA:DNA duplexes is largely insensitive to ionic strength, it is significantly lower than that of DNA:DNA duplexes having the same number and sequence of base pairing interactions. The temperature dependence of PNA:DNA kinetic rate constants indicate a significant enthalpy barrier to duplex dissociation, and to a lesser extent, duplex formation. This investigation into the kinetics of PNA:DNA hybridization provides a framework towards better understanding and design of PNA sequences for future applications. 

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2. Kinetics and dynamics of oligonucleotide hybridization

   https://doi.org/10.1038/s41570-025-00704-8  

   Ashwood, Brennan; Tokmakoff, Andrei (May 2025, Nature Reviews Chemistry)

   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 and 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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3. Non-equilibrium structural dynamics of supercoiled DNA plasmids exhibits asymmetrical relaxation

   https://doi.org/10.1093/nar/gkac101  

   Shaheen, Cynthia; Hastie, Cameron; Metera, Kimberly; Scott, Shane; Zhang, Zhi; Chen, Sitong; Gu, Gracia; Weber, Lisa; Munsky, Brian; Kouzine, Fedor; et al (February 2022, Nucleic Acids Research)

   Abstract Many cellular processes occur out of equilibrium. This includes site-specific unwinding in supercoiled DNA, which may play an important role in gene regulation. Here, we use the Convex Lens-induced Confinement (CLiC) single-molecule microscopy platform to study these processes with high-throughput and without artificial constraints on molecular structures or interactions. We use two model DNA plasmid systems, pFLIP-FUSE and pUC19, to study the dynamics of supercoiling-induced secondary structural transitions after perturbations away from equilibrium. We find that structural transitions can be slow, leading to long-lived structural states whose kinetics depend on the duration and direction of perturbation. Our findings highlight the importance of out-of-equilibrium studies when characterizing the complex structural dynamics of DNA and understanding the mechanisms of gene regulation. 

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4. Understanding DNA interactions in crowded environments with a coarse-grained model

   https://doi.org/10.1093/nar/gkaa854  

   Hong, Fan; Schreck, John S; Šulc, Petr (October 2020, Nucleic Acids Research)

   null (Ed.)

   Abstract Nucleic acid interactions under crowded environments are of great importance for biological processes and nanotechnology. However, the kinetics and thermodynamics of nucleic acid interactions in a crowded environment remain poorly understood. We use a coarse-grained model of DNA to study the kinetics and thermodynamics of DNA duplex and hairpin formation in crowded environments. We find that crowders can increase the melting temperature of both an 8-mer DNA duplex and a hairpin with a stem of 6-nt depending on the excluded volume fraction of crowders in solution and the crowder size. The crowding induced stability originates from the entropic effect caused by the crowding particles in the system. Additionally, we study the hybridization kinetics of DNA duplex formation and the formation of hairpin stems, finding that the reaction rate kon is increased by the crowding effect, while koff is changed only moderately. The increase in kon mostly comes from increasing the probability of reaching a transition state with one base pair formed. A DNA strand displacement reaction in a crowded environment is also studied with the model and we find that rate of toehold association is increased, with possible applications to speeding up strand displacement cascades in nucleic acid nanotechnology. 

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5. Hybridization kinetics of out-of-equilibrium mixtures of short RNA oligonucleotides

   https://doi.org/10.1093/nar/gkac784  

   Todisco, Marco; Szostak, Jack W. (September 2022, Nucleic Acids Research)

   Abstract Hybridization and strand displacement kinetics determine the evolution of the base paired configurations of mixtures of oligonucleotides over time. Although much attention has been focused on the thermodynamics of DNA and RNA base pairing in the scientific literature, much less work has been done on the time dependence of interactions involving multiple strands, especially in RNA. Here we provide a study of oligoribonucleotide interaction kinetics and show that it is possible to calculate the association, dissociation and strand displacement rates displayed by short oligonucleotides (5nt–12nt) that exhibit no expected secondary structure as simple functions of oligonucleotide length, CG content, ΔG of hybridization and ΔG of toehold binding. We then show that the resultant calculated kinetic parameters are consistent with the experimentally observed time dependent changes in concentrations of the different species present in mixtures of multiple competing RNA strands. We show that by changing the mixture composition, it is possible to create and tune kinetic traps that extend by orders of magnitude the typical sub-second hybridization timescale of two complementary oligonucleotides. We suggest that the slow equilibration of complex oligonucleotide mixtures may have facilitated the nonenzymatic replication of RNA during the origin of life. 

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