Sarkar Bidyut, Ishii Kunihiko, Tahara Tahei
Molecular Spectroscopy Laboratory , RIKEN , 2-1 Hirosawa , Wako , Saitama 351-0198 , Japan.
Ultrafast Spectroscopy Research Team , RIKEN Center for Advanced Photonics (RAP) , 2-1 Hirosawa , Wako , Saitama 351-0198 , Japan.
J Phys Chem Lett. 2019 Sep 19;10(18):5536-5541. doi: 10.1021/acs.jpclett.9b01513. Epub 2019 Sep 5.
The single-molecule Förster resonance energy transfer (smFRET) technique is widely used for studying conformational dynamics of biopolymers. However, smFRET requires double dye labeling and is usually utilized for detecting dynamics on slow time scales (≳ milliseconds). In this Letter, we report dynamic-quenching two-dimensional fluorescence lifetime correlation spectroscopy (DQ 2D FLCS) that can elucidate the microsecond conformational dynamics of biopolymers with only single dye labeling. In DQ 2D FLCS, the difference in solvent accessibility of the labeled dye makes the fluorescence lifetime different, which is used for distinguishing different conformers. By applying DQ 2D FLCS to a singly labeled DNA hairpin, we successfully detect microsecond interconversion dynamics between the open and closed forms and evaluate the state-specific solvent accessibility of each form with Stern-Volmer analysis. Because DQ 2D FLCS is sensitive to the local structural change, it is complementary to FRET-based 2D FLCS and thus is a new, powerful tool for studying structural dynamics of biopolymers.
单分子荧光共振能量转移（smFRET）技术被广泛用于研究生物聚合物的构象动力学。然而，smFRET需要进行双染料标记，并且通常用于检测慢时间尺度（≳毫秒）上的动力学。在本信函中，我们报道了动态猝灭二维荧光寿命相关光谱（DQ 2D FLCS），它仅通过单染料标记就能阐明生物聚合物的微秒级构象动力学。在DQ 2D FLCS中，标记染料的溶剂可及性差异使得荧光寿命不同，这被用于区分不同的构象体。通过将DQ 2D FLCS应用于单标记的DNA发夹结构，我们成功检测到了开放和闭合形式之间的微秒级相互转换动力学，并通过Stern-Volmer分析评估了每种形式的状态特异性溶剂可及性。由于DQ 2D FLCS对局部结构变化敏感，它与基于FRET的2D FLCS互补，因此是研究生物聚合物结构动力学的一种新型强大工具。
