Single-Molecule Kinetics Reveal Cation-Promoted DNA Duplex Formation Through Ordering of Single-Stranded Helices
Single-Molecule Kinetics Reveal Cation-Promoted DNA Duplex Formation Through Ordering of Single-Stranded Helices
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DOI：
10.1016/j.bpj.2013.05.061
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发表时间：
2013-08-06
影响因子：
3.4
通讯作者：
Nesbitt, David J.
中科院分区：
文献类型：
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作者：
Dupuis, Nicholas F.;Holmstrom, Erik D.;Nesbitt, David J.
在这项工作中，在单分子水平上研究了短的，完全互补的寡核苷酸的动力学。长度为6- 9bp的构建体在2个数量级的时间内，在正向（k(on)，结合）和反向（k(off)，解离）过程中都表现出单指数动力学。结合的双分子速率常数对双链中碱基对的数量很弱敏感，在9 bp (k(on)' = 2.1(1) × 10(6) M（-1)s(-1)）和6 bp (k(on)' = 5.0(1) × 10（6) M-1 s(-1)）序列之间增加2.5倍。然而，与之形成鲜明对比的是，解离速率常数对序列长度呈指数级敏感，在相同的9 bp (k（off) = 0.024 s(-1)）至6 bp (k（off) = 14 s(-1)）范围内变化近600倍。更详细地探讨了8bp序列，并测量了k（on）和k（off）对NaCl的依赖性。有趣的是，在[NaCl] = 25 mM和1 M之间，k（on）增加了40倍(k(on) = 0.10(1) s（-1）到4.0(4)s(-1))，而在相同的条件范围内，k（off）减少了4倍(0.72(3)s（-1）到0.17（7)s(-1)）。因此，平衡常数（K-eq）增加了大约160，主要是由于缔合速率km的变化。最后，温度相关的测量结果表明，[NaCl]的增加降低了双相形成的总体放热性（δ δ H度>），尽管其减少的量小于熵罚的减少量（-T δ δ S度< 0）。这种熵成本的降低是由于阳离子促进了两个单链物种的预先排序，从而降低了结合的自由能垒，从而加快了双链形成的速度。
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) x 10(6) M(-1)s(-1)) and 6 bp (k(on)' = 5.0(1) x 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 (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(on) increases 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-off decreases 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 approximate to 160, largely due to changes in the association rate, km. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (Delta Delta H degrees > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (-T Delta Delta S degrees < 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.