Foote Alexander K, Ishii Kunihiko, Cullinane Brendan, Tahara Tahei, Goldsmith Randall H
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.
Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
ACS Phys Chem Au. 2024 May 29;4(4):408-419. doi: 10.1021/acsphyschemau.3c00066. eCollection 2024 Jul 24.
Quantifying the rapid conformational dynamics of biological systems is fundamental to understanding the mechanism. However, biomolecules are complex, often containing static and dynamic heterogeneity, thus motivating the use of single-molecule methods, particularly those that can operate in solution. In this study, we measure microsecond conformational dynamics of solution-phase DNA hairpins at the single-molecule level using an anti-Brownian electrokinetic (ABEL) trap. Different conformational states were distinguished by their fluorescence lifetimes, and kinetic parameters describing transitions between these states were determined using two-dimensional fluorescence lifetime correlation (2DFLCS) analysis. Rather than combining fluorescence signals from the entire data set ensemble, long observation times of individual molecules allowed ABEL-2DFLCS to be performed on each molecule independently, yielding the underlying distribution of the system's kinetic parameters. ABEL-2DFLCS on the DNA hairpins resolved an underlying heterogeneity of fluorescence lifetimes and provided signatures of two-state exponential dynamics with rapid (<millisecond) transition times between states without observation of the substantially stretched exponential kinetics that had been observed in previous measurements on diffusing molecules. Numerical simulations were performed to validate the accuracy of this technique and the effects the underlying heterogeneity has on the analysis. Finally, ABEL-2DFLCS was performed on a mixture of hairpins and used to resolve their kinetic data.
量化生物系统快速的构象动力学对于理解其机制至关重要。然而，生物分子很复杂，常常包含静态和动态的异质性，因此促使人们使用单分子方法，尤其是那些能在溶液中操作的方法。在本研究中，我们使用抗布朗电动（ABEL）阱在单分子水平测量溶液相DNA发夹的微秒级构象动力学。通过荧光寿命区分不同的构象状态，并使用二维荧光寿命相关（2DFLCS）分析确定描述这些状态之间转变的动力学参数。与将整个数据集总体的荧光信号合并不同，对单个分子的长时间观察使得ABEL - 2DFLCS能够独立地对每个分子进行，从而得出系统动力学参数的潜在分布。对DNA发夹进行的ABEL - 2DFLCS解析了荧光寿命的潜在异质性，并提供了双态指数动力学的特征，其状态之间的转变时间很快（<毫秒），而未观察到在先前对扩散分子的测量中所观察到的明显拉伸指数动力学。进行了数值模拟以验证该技术的准确性以及潜在异质性对分析的影响。最后，对发夹混合物进行了ABEL - 2DFLCS并用于解析其动力学数据。
