Hartmann Andreas, Krainer Georg, Schlierf Michael
B CUBE, Center for Molecular Bioengineering, Technische Universität Dresden, Dresden 01307, Germany.
Molecules. 2014 Sep 3;19(9):13735-54. doi: 10.3390/molecules190913735.
Changes in molecular conformations are one of the major driving forces of complex biological processes. Many studies based on single-molecule techniques have shed light on conformational dynamics and contributed to a better understanding of living matter. In particular, single-molecule FRET experiments have revealed unprecedented information at various time scales varying from milliseconds to seconds. The choice and the attachment of fluorophores is a pivotal requirement for single-molecule FRET experiments. One particularly well-studied millisecond conformational change is the opening and closing of DNA hairpin structures. In this study, we addressed the influence of base- and terminal-labeled fluorophores as well as the fluorophore DNA interactions on the extracted kinetic information of the DNA hairpin. Gibbs free energies varied from ∆G0 = -3.6 kJ/mol to ∆G0 = -0.2 kJ/mol for the identical DNA hairpin modifying only the labeling scheme and design of the DNA sample. In general, the base-labeled DNA hairpin is significantly destabilized compared to the terminal-labeled DNA hairpin and fluorophore DNA interactions additionally stabilize the closed state of the DNA hairpin. Careful controls and variations of fluorophore attachment chemistry are essential for a mostly undisturbed measurement of the underlying energy landscape of biomolecules.
分子构象的变化是复杂生物过程的主要驱动力之一。许多基于单分子技术的研究揭示了构象动力学，并有助于更好地理解生命物质。特别是，单分子荧光共振能量转移（FRET）实验在从毫秒到秒的各种时间尺度上揭示了前所未有的信息。荧光团的选择和连接是单分子FRET实验的关键要求。一个经过充分研究的毫秒级构象变化是DNA发夹结构的打开和关闭。在本研究中，我们探讨了碱基标记和末端标记的荧光团以及荧光团与DNA的相互作用对提取的DNA发夹动力学信息的影响。对于仅改变DNA样品标记方案和设计的相同DNA发夹，吉布斯自由能从∆G0 = -3.6 kJ/mol变化到∆G0 = -0.2 kJ/mol。一般来说，与末端标记的DNA发夹相比，碱基标记的DNA发夹明显不稳定，并且荧光团与DNA的相互作用进一步稳定了DNA发夹的闭合状态。仔细控制和改变荧光团连接化学对于大多数情况下不受干扰地测量生物分子的潜在能量景观至关重要。
