Department of Chemistry, Wayne State University, Detroit, Michigan, USA.
Biophys J. 2010 Sep 22;99(6):1925-31. doi: 10.1016/j.bpj.2010.07.019.
To assemble into functional structures, biopolymers search for global minima through their folding potential energy surfaces to find the native conformation. However, this process can be hindered by the presence of kinetic traps. Here, we present a new single-molecule technique, termed laser-assisted single-molecule refolding (LASR), to characterize kinetic traps at the single-molecule level. LASR combines temperature-jump kinetics and single-molecule spectroscopy. We demonstrate the use of LASR to measure single-molecule DNA melting curves with ∼1°C accuracy and to determine the activation barrier of a model kinetic trap. We also show how LASR, in combination with mutagenesis, can be used to estimate the yields of competing pathways, as well as to generate and characterize transient, unstable complexes.
为了组装成功能性结构，生物聚合物通过它们的折叠势能表面搜索全局最小值，以找到天然构象。然而，这个过程可能会受到动力学陷阱的阻碍。在这里，我们提出了一种新的单分子技术，称为激光辅助单分子重折叠（LASR），以在单分子水平上表征动力学陷阱。LASR 结合了温度跃变动力学和单分子光谱学。我们展示了如何使用 LASR 以约 1°C 的精度测量单分子 DNA 熔解曲线，并确定模型动力学陷阱的激活势垒。我们还展示了 LASR 如何与诱变结合使用，以估计竞争途径的产率，以及生成和表征瞬态、不稳定的复合物。
