Single-Molecule Titration in a Protein Nanoreactor Reveals the Protonation/Deprotonation Mechanism of a C:C Mismatch in DNA,Journal of the American Chemical Society

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Single-Molecule Titration in a Protein Nanoreactor Reveals the Protonation/Deprotonation Mechanism of a C:C Mismatch in DNA
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2018-03-21 , DOI: 10.1021/jacs.8b00593
Hang Ren ₁ , Cameron G. Cheyne ₁ , Aaron M. Fleming ₁ , Cynthia J. Burrows ₁ , Henry S. White ₁

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Measurement of single-molecule reactions can elucidate microscopic mechanisms that are often hidden from ensemble analysis. Herein, we report the acid-base titration of a single DNA duplex confined within the wild-type α-hemolysin (α-HL) nanopore for up to 3 h, while monitoring the ionic current through the nanopore. Modulation between two states in the current-time trace for duplexes containing the C:C mismatch in proximity to the latch constriction of α-HL is attributed to the base flipping of the C:C mismatch. As the pH is lowered, the rate for the C:C mismatch to flip from the intra-helical state to the extra-helical state ( kintra-extra) decreases, while the rate for base flipping from the extra-helical state to the intra-helical state ( kextra-intra) remains unchanged. Both kintra-extra and kextra-intra are on the order of 1 × 10-2 s-1 to 1 × 10-1 s-1 and remain stable over the time scale of the measurement (several hours). Analysis of the pH-dependent kinetics of base flipping using a hidden Markov kinetic model demonstrates that protonation/deprotonation occurs while the base pair is in the intra-helical state. We also demonstrate that the rate of protonation is limited by transport of H+ into the α-HL nanopore. Single-molecule kinetic isotope experiments exhibit a large kinetic isotope effect (KIE) for kintra-extra ( kH/ kD ≈ 5) but a limited KIE for kextra-intra ( kH/ kD ≈ 1.3), supporting our model. Our experiments correspond to the longest single-molecule measurements performed using a nanopore, and demonstrate its application in interrogating mechanisms of single-molecule reactions in confined geometries.

中文翻译：

蛋白质纳米反应器中的单分子滴定揭示 DNA 中 C:C 错配的质子化/去质子化机制

单分子反应的测量可以阐明通常隐藏在集合分析中的微观机制。在此，我们报告了限制在野生型 α-溶血素 (α-HL) 纳米孔内的单个 DNA 双链体的酸碱滴定长达 3 小时，同时监测通过纳米孔的离子电流。对于包含 C:C 失配接近 α-HL 锁存收缩的双工，当前时间轨迹中两个状态之间的调制归因于 C:C 失配的基极翻转。随着 pH 值降低，C:C 错配从内螺旋状态翻转到外螺旋状态 (kintra-extra) 的速率降低，而碱基从外螺旋状态翻转到内螺旋状态的速率-螺旋状态（kextra-intra）保持不变。kintra-extra 和 kextra-intra 都在 1 × 10-2 s-1 到 1 × 10-1 s-1 的数量级上，并且在测量的时间尺度（几个小时）内保持稳定。使用隐马尔可夫动力学模型分析碱基翻转的 pH 依赖性动力学表明，当碱基对处于螺旋内状态时，会发生质子化/去质子化。我们还证明质子化率受到 H+ 运输到 α-HL 纳米孔的限制。单分子动力学同位素实验对 kintra-extra ( kH/ kD ≈ 5) 表现出较大的动力学同位素效应 (KIE)，但对 kextra-intra ( kH/ kD ≈ 1.3) 表现出有限的 KIE，支持我们的模型。我们的实验对应于使用纳米孔进行的最长单分子测量，并证明了其在研究受限几何结构中单分子反应机制中的应用。

更新日期：2018-03-21

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