Long Xi, Parks Joseph W, Stone Michael D
Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA, USA.
Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA, USA; Center for Molecular Biology of RNA, University of California Santa Cruz, Santa Cruz, CA, USA.
Methods. 2016 Aug 1;105:16-25. doi: 10.1016/j.ymeth.2016.06.009. Epub 2016 Jun 15.
Many enzymes promote structural changes in their nucleic acid substrates via application of piconewton forces over nanometer length scales. Magnetic tweezers (MT) is a single molecule force spectroscopy method widely used for studying the energetics of such mechanical processes. MT permits stable application of a wide range of forces and torques over long time scales with nanometer spatial resolution. However, in any force spectroscopy experiment, the ability to monitor structural changes in nucleic acids with nanometer sensitivity requires the system of interest to be held under high degrees of tension to improve signal to noise. This limitation prohibits measurement of structural changes within nucleic acids under physiologically relevant conditions of low stretching forces. To overcome this challenge, researchers have integrated a spatially sensitive fluorescence spectroscopy method, single molecule-FRET, with MT to allow simultaneous observation and manipulation of nanoscale structural transitions over a wide range of forces. Here, we describe a method for using this hybrid instrument to analyze the mechanical properties of nucleic acids. We expect that this method for analysis of nucleic acid structure will be easily adapted for experiments aiming to interrogate the mechanical responses of other biological macromolecules.
许多酶通过在纳米长度尺度上施加皮牛顿力来促进其核酸底物的结构变化。磁镊（MT）是一种单分子力谱方法，广泛用于研究此类机械过程的能量学。磁镊允许在长时间尺度上以纳米空间分辨率稳定地施加广泛的力和扭矩。然而，在任何力谱实验中，要以纳米灵敏度监测核酸的结构变化，就需要将感兴趣的系统置于高度张力下以提高信噪比。这一限制使得在生理相关的低拉伸力条件下无法测量核酸内部的结构变化。为了克服这一挑战，研究人员将一种空间敏感的荧光光谱方法——单分子荧光共振能量转移（single molecule-FRET）与磁镊相结合，以便在广泛的力范围内同时观察和操纵纳米级结构转变。在这里，我们描述了一种使用这种混合仪器分析核酸力学性质的方法。我们预计，这种核酸结构分析方法将很容易适用于旨在探究其他生物大分子力学响应的实验。
