Wang Ying, Sischka Andy, Walhorn Volker, Tönsing Katja, Anselmetti Dario
Experimental Biophysics, Physics Faculty, Bielefeld University, Bielefeld, Germany.
Experimental Biophysics, Physics Faculty, Bielefeld University, Bielefeld, Germany.
Biophys J. 2016 Oct 18;111(8):1604-1611. doi: 10.1016/j.bpj.2016.08.042.
Fluorescent DNA dyes are broadly used in many biotechnological applications for detecting and imaging DNA in cells and gels. Their binding alters the structural and nanomechanical properties of DNA and affects the biological processes that are associated with it. Although interaction modes like intercalation and minor groove binding already have been identified, associated mechanic effects like local elongation, unwinding, and softening of the DNA often remain in question. We used magnetic tweezers to quantitatively investigate the impact of three DNA-binding dyes (YOYO-1, DAPI, and DRAQ5) in a concentration-dependent manner. By extending and overwinding individual, torsionally constrained, nick-free dsDNA molecules, we measured the contour lengths and molecular forces that allow estimation of thermodynamic and nanomechanical binding parameters. Whereas for YOYO-1 and DAPI the binding mechanisms could be assigned to bis-intercalation and minor groove binding, respectively, DRAQ5 exhibited both binding modes in a concentration-dependent manner.
荧光DNA染料广泛应用于许多生物技术应用中，用于检测细胞和凝胶中的DNA并对其进行成像。它们的结合会改变DNA的结构和纳米力学性质，并影响与之相关的生物学过程。尽管诸如嵌入和小沟结合等相互作用模式已经得到确认，但DNA的局部伸长、解旋和软化等相关力学效应往往仍存在疑问。我们使用磁镊以浓度依赖的方式定量研究了三种DNA结合染料（YOYO-1、DAPI和DRAQ5）的影响。通过拉伸和过度缠绕单个、无扭转约束、无缺口的双链DNA分子，我们测量了轮廓长度和分子力，从而可以估算热力学和纳米力学结合参数。对于YOYO-1和DAPI，结合机制分别可归因于双嵌入和小沟结合，而DRAQ5在浓度依赖的方式下表现出两种结合模式。
