Influence of thermodynamically unfavorable secondary structures on DNA hybridization kinetics

Abstract

Nucleic acid secondary structure plays an important role in nucleic acid-nucleic acid recognition/hybridization processes, and is also a vital consideration in DNA nanotechnology. Al-though the influence of stable secondary structures on hybridization kinetics has been characterized, unstable secondary structures, which show positive AG with self-folding, can also form, and their effects have not been systematically investigated. Such thermodynamically unfavorable secondary structures should not be ignored in DNA hybridization kinetics, especially under isothermal conditions. Here, we report that positive ΔG° secondary structures can change the hybridization rate by two-orders of magnitude, despite the fact that their hybridization obeyed second-order reaction kinetics. The temperature dependence of hybridization rates showed non-Arrhenius behavior; thus, their hybridization is considered to be nucleation limited. We derived a model describing how ΔG° positive secondary structures affect hybridization kinetics in stopped-flow experiments with 47 pairs of oligonucleotides. The calculated hybridization rates, which were based on the model, quantitatively agreed with the experimental rate constant.