A Single Molecule Kinetic and Thermodynamic Approach to Oligonucleotide Duplex Formation

Abstract

The formation of the canonical double stranded helix of DNA and RNA, through oligonucleotide hybridization, is a ubiquitous process in biology. Here we describe a single molecule fluorescence resonance energy transfer (smFRET) study that probes the formation and dissociation of a short (6-9 base pairs) double stranded DNA duplex. Kinetic measurements on this construct reveal that rates of duplex formation are not diffusion limited. In fact, the rate constants of formation are 2-3 orders of magnitude slower than estimated with a simple diffusion model. Subsequently, the duplex formation/dissociation (kon and koff) rates were measured as a function of Na+ concentration and both were found to change in a sigmoidal fashion with [Na+]. The measured values of koff decreased by a factor of four (0.7(2) s-1 to 0.14(5) s-1) between 25 mM and 1 M Na+. Surprisingly, kon was found to be much more sensitive to the [Na+], increasing by more than a factor of 40 (0.6(6) s-1 to 4.4(6) s-1) over the same range. These changes combine give a >100 fold change in the unimolecular equilibrium constant (Keq). Finally, the temperature dependence of Keq was used to dissect the free energy of duplex formation (ΔG°) into its enthalpic (ΔH°) and entropic (ΔS°) components. While ΔH° remains negative under all conditions, increasing the [Na+] reduced the magnitudes of both ΔH° and ΔS°. Given that ΔG° decreases with increasing [Na+], the magnitude of ΔS° must decrease faster than the unfavorable reduction in the magnitude of ΔH°. These insights highlight the dynamic nature of DNA duplexes on the time scale (seconds to minutes) of many cellular processes. Additionally, these data may aid in developing and evaluating new methods for modeling the thermodynamics of DNA duplex formation.