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

Abstract

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 (kon, association) and reverse (koff, 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) × 10(6) M(-1) s(-1)) and 6 bp (k'on = 5.0(1) × 10(6) 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 (koff = 0.024 s(-1)) to 6 bp (koff = 14 s(-1)) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, kon increases by >40-fold (kon = 0.10(1) s(-1) to 4.0(4) s(-1) between [NaCl] = 25 mM and 1 M), whereas in contrast, koff decreases by fourfold (0.72(3) s(-1) to 0.17(7) s(-1)) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. 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.

Electrostatic melting in a single-molecule field-effect transistor with applications in genomic identification

“…A relatively linear target concentration dependence is observed, as expected from the diffusion-limited process. At a target concentration of 100 nM, however, k hyb does not appreciably increase because under these conditions, k hyb is reaction-limited, an observation consistent with ensemble3132 and single-molecule fluorescence resonance energy transfer studies2. In addition, k hyb at the 1 and 10 nM concentrations are expected to be accelerated compared to the 100 nM concentration, since the contribution of intermediate pathways to k hyb becomes insignificant at these lower concentrations, again consistent with ensemble studies133334.…”

Understanding Nucleic Acid–Ion Interactions

“…Further complicating the analysis of nucleic acid–ion behaviors and energetics is the presence of large ensembles of conformations of the nucleic acids of interest. Thus, in addition to determining the nucleic acid–ion energetics of one nucleic acid conformation, any modeling effort must repeat this calculation many times, for each of the nucleic acid conformations that represent the overall ensemble of conformers present (39–43). …”

Kinetic fingerprinting to identify and count single nucleic acids

“…1b,c). Because the kinetics of exchange for probes of ~6–12 nt are highly sensitive to the number of complementary bases between the probe and target 7,10,11 , varying the length of the probe allows fine-tuning of the kinetic behavior to improve specificity of detection. For the probes used in this study, kinetics of binding and dissociation were found to be more closely correlated to probe length than to the melting temperature of the duplex (Supplementary Fig.…”

“…Because the lifetime of a short DNA duplex increases as an approximately exponential function of the number of base pairs 7,10,11 , we reasoned that SiMREPS might be used to achieve excellent single-base discrimination. To test this hypothesis, we used a single fluorescent probe to discriminate between two let-7 family members, hsa-let-7a and hsa-let-7c .…”