基于纳米操纵和单分子成像及分析技术的DNA与DNase I相互作用的表面机械生化过程和动力学研究

Mechanochemical activation and kinetics in DNase I digestion revealed by nanomanipulation and single-molecule imaging & analysis

本文采用了"多步的改进的动态分子梳"纳米操纵技术(multi-step modified dynamic molecular combing,MMDMC)实现样品制备获得不同拉伸程度单个DNA分子,利用原子力显微镜重定位成像和单分子分析,对反应前后不同拉伸程度DNA分子的DNase I酶切产生缺口情况进行统计,在一定拉伸范围内讨论了在表面上DNaseI酶切反应速率变化的动力学信息。该不同拉伸程度的DNA与DNase I相互作用的表面机械生化过程对于生物分子多次反复相互作用研究体系具有代表性意义。

How the mechanochemistry controls a reaction through mechanical deformation of a reagent by an external force is a fundamental and currently hot issue in biochemistry and biophysics. It has been theoretically predicted that the tension of only a few piconewtons acting on single biomolecules, where the distinct states are not blurred by bulk averaging, should lead to totally different mechanochemical processes. Whether an applied force affects enzymes acting on the mechanically stretched DNA molecules and subsequently cleavage rate or not, is a focus with its significance in biology. Herein, we showed our results on the kinetic profiles of DNase I digestion of individual DNA molecules that were mechanically stretched by using a method of nanomanipulation termed as ＂multi-step modified dynamic molecular combing＂ （MMDMC）. The atomic force microscopy （AFM） imaging ＆ analysis were used to measure the cleaved gaps on these DNA molecules with different stretching extents before and after the digestion. Detailed kinetic profiles of the reaction have been revealed at the lower tension, and a meehanochemical activation in DNase I digestion has been discovered. Crystallographic studies on the DNase I-DNA complex had indicated that the stretching resulted in wider minor grooves on DNA molecule, which may be more accessible for the enzyme to recognize and afterward cut the DNA. We believe the possibility of further explorations in combination with some other results out from different nanotechniques, such as magnetic tweezers, optical/laser tweezers, and molecular dynamics （MD） simulations based on the X-ray structural data and crystallographic studies.