氟中毒机制单分子研究

High dose intake of fluoride from contaminated drinking water leads to fluorosis. Around half of the population in Guizhou province suffer.from fluoroisis, but the mechanism of fluoride toxicity remains elusive. A fluoride-responsive riboswitch class was identified by analyzing a group of noncoding RNA structures that carry a conserved domain called the crcB motif. crcB motif RNAs are located upstream of genes encoding proteins of diverse functions and presumably regulate these genes. The conformation of crcB motif can be changed by adding fluoride ion, but exclusive to other halogen ions. Fluoride ion serves as potential acceptor of hydrogen bond, the exitence of the direct formation of hydrogen bond beteween fluoride ion and the base of DNA . Based on the hairpin structure, effect of fluoride on the stability.of hairpin structure will be detected by magnetic tweezers, and then the association constant between fluoride ion and the base in oligonucleotide designed according to the hairpin structure can be detected by ITC experiment. Finally a realition between the association constant and the binding energy of hydrogen bond can be defined based on statistical machanics, by comaring it with the association constant obtained in ITC experiment, the binding energy can be obtained definitely, so that the exsitence of hydrogen bond can be identified.

氟中毒分布广，且致残率高，贵州省为重病区，病区人口接近总人口二分之一，确切致病机制仍不清楚。最近实验发现一类调控氟离子转运的CLC阴离子输运蛋白， 为一类反向转运体，其表达由含有保守区域crcB motif非编码RNA调控。crcB motif位于编码多功能蛋白基因上游，编码膜蛋白。不同于其它卤族离子，氟离子显著影响crcB motif构象，进而影响基因表达。氟离子为潜在氢键受体，其能否与碱基形成氢键，进而影响crcB motif构象，尚不清楚。基于crcB motif 亚结构特征，构建DNA及RNA hairpin结构，利用磁镊子确定氟化物对hairpin结构稳定性影响一般规律；在此基础上，利用等温滴定量热法确定氟离子与寡聚核苷酸链之间结合常数；基于统计力学方法确定结合常数与结合能之间定量关系，结合实验确定的结合常数，确定氟离子与碱基之间结合能，判定氟离子与碱基之间是否形成氢键。

按照预定研究计划完成国家自然科学基金“氟中毒机制单分子研究”的研究任务，项目结合单分子实验、等温滴定量热实验、分子动力学模拟等方法系统地研究了氟化物对DNA hairpin结构稳定性的影响，取得以下主要结果， (1)搭建单分子磁镊平台，利用打开概率、转变速率分析不同氟离子浓度条件下的hairpin结构转变的动力学信息，DNA hairpin结构的临界力随着氟离子浓度增加而逐渐减少； (2) 利用统计力学方法建立了结合常数与结合能之间的关系，将其与ITC实验测量的的寡聚核苷酸链结合常数结合，确定氢键能量，利用该方法证实氟离子与寡聚核苷酸链间未形成氢键； (3) 氟离子浓度增加导致寡聚核苷酸链的Zeta电位逐渐降低，导致DNA hairpin结构失稳。分子动力学结果表明，氟离子相对于寡聚核苷酸链的径向分布比氯离子的径向分布要广，导致Zeta电位降低； (4)拓展oxDNA模型研究大分子拥挤效应，模拟结果与单分子实验结果对比表明PEG分子引起的大分子拥挤效应对DNA hairpin结构稳定性影响是通过排斥体积效应实现的。