Single-Molecule Kinetics Reveal Cation-Promoted DNA Duplex Formation Through Ordering of Single-Stranded Helices

In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (k_on, association) and reverse (k_off, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k′_on = 2.1(1) × 10⁶ M⁻¹ s⁻¹) and 6 bp (k′_on = 5.0(1) × 10⁶ M⁻¹ s⁻¹) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (k_off = 0.024 s⁻¹) to 6 bp (k_off = 14 s⁻¹) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of k_on and k_off is measured. Interestingly, k_onincreases by >40-fold (k_on = 0.10(1) s⁻¹ to 4.0(4) s⁻¹ between NaCl] = 25 mM and 1 M), whereas in contrast, k_offdecreases by fourfold (0.72(3) s⁻¹ to 0.17(7) s⁻¹) over the same range of conditions. Thus, the equilibrium constant (K_eq) increases by ≈160, largely due to changes in the association rate, k_on. Finally, temperature-dependent measurements reveal that increased NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (−TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.