Cullinane Brendan, Ishii Kunihiko, Kaur Simi, Tahara Tahei, Goldsmith Randall H
Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, Wisconsin 53705, United States.
Molecular Spectroscopy Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
J Am Chem Soc. 2025 Apr 23;147(16):13101-13107. doi: 10.1021/jacs.4c17471. Epub 2025 Apr 8.
DNA hairpins are a model system for biomolecule folding as well as key structures in biology and nanotechnology. However, limitations in traditional solution-phase spectroscopy shorten the window of observable kinetics and cannot account for static heterogeneity. Here, we show that the application of two-dimensional fluorescence lifetime correlation spectroscopy (2DFLCS) to a solution-phase molecule trapped in an anti-Brownian electrokinetic (ABEL) trap bypasses those limitations, enabling kinetic analysis of the dynamics of single solution-phase molecules on a broad range of time scales down to microseconds. The analysis unambiguously shows that DNA hairpin folding proceeds via a three-state system, where hairpins fold initially on the scale of tens to hundreds of microseconds from a random coil to a partially closed intermediate and then form a stable fully closed state.
DNA发夹是生物分子折叠的模型系统，也是生物学和纳米技术中的关键结构。然而，传统溶液相光谱学的局限性缩短了可观测动力学的窗口，并且无法解释静态异质性。在这里，我们表明，将二维荧光寿命相关光谱学（2DFLCS）应用于捕获在反布朗电动（ABEL）阱中的溶液相分子，可以绕过这些限制，从而能够在低至微秒的广泛时间尺度上对单个溶液相分子的动力学进行动力学分析。分析明确表明，DNA发夹折叠通过三态系统进行，其中发夹最初在数十至数百微秒的时间尺度上从无规卷曲折叠成部分闭合的中间体，然后形成稳定的完全闭合状态。
