Department of Chemistry and Biochemistry , Kent State University , Kent , Ohio 44242 , United States.
Anal Chem. 2018 Mar 6;90(5):3205-3210. doi: 10.1021/acs.analchem.7b04661. Epub 2018 Feb 15.
Concentration jumps for kinetics measurement remain a challenge for single-molecule techniques, which have demonstrated superior signal-to-noise levels compared to ensemble average approaches. Currently, all concentration jumps use mixing strategies. Here, we report a simple and drastically different jump strategy by rapid transportation of molecules between two side-by-side laminar streams in 80 ms. This allowed us to measure the lifetime of bioactive DNA i-motif structures at physiological pH without force. We placed a human telomeric i-motif inside a DNA hairpin-based mechanical reporter. Since the folded or unfolded state of the hairpin correlates with that of the i-motif, by recording hairpin transitions, a half-life of ∼3 s was found for the DNA i-motif at neutral pH without force. Such a lifetime is sufficient for i-motif to interact with proteins to modulate cellular processes. We anticipate this concentration jump offers a generic platform to investigate single-molecule kinetics.
浓度跃变对于动力学测量仍然是一个挑战，单分子技术相比平均集合方法具有更高的信噪比。目前，所有的浓度跃变都使用混合策略。在这里，我们报告了一种简单而截然不同的跃变策略，通过在 80 毫秒内将分子快速输送到两个并排的层流之间。这使我们能够在没有力的情况下测量生理 pH 值下生物活性 DNA i-motif 结构的寿命。我们将人类端粒 i-motif 置于基于 DNA 发夹的机械报告器内。由于发夹的折叠或展开状态与 i-motif 的状态相关，通过记录发夹的转变，我们发现 DNA i-motif 在中性 pH 值下没有力的半衰期约为 3 秒。这样的寿命足以使 i-motif 与蛋白质相互作用以调节细胞过程。我们预计这种浓度跃变将为研究单分子动力学提供一个通用平台。
