摘要
目的探讨乙醇脱氢核酶ribox02与NAD^+、NADH特异性结合的位点以及结合力性质。方法通过选择性2-OH酰基化学探测法(selective 2-hydroxyl acylation analyzed by primer extension,SHAPE)探测ribox02的二级结构;利用等温滴定量热法(isothermal titratio calorimetry,ITC)进一步测定ribox02与NADH、NAD^+的亲和力强度及作用力性质;通过紫外交联法精确定位NAD^+、NADH与ribox02 RNA的特异性结合位点。结果直接解析ribox02的二级结构,并发现核酶ribox02与NADH、NAD^+的相互作用力为范德华力或氢键作用,且ribox02与NADH的相互作用力较之与NAD^+更为强烈,ribox02与NADH、NAD^+的特异性结合位点为23G、24U、25U、38U、72U、73U、74U、75G和95G。结论通过ribox02的二级结构及其与NAD^+、NADH的相互作用揭示了核酶ribox02与NAD^+/NADH特异性结合位点以及结合力性质。
Objective To explore the affinity and specific binding sites between alcohol dehydrogenase ribozyme ribox02 and NAD^＋/NADH. Methods We analyzed the secondary structure of ribox02 by the selective 2 hydroxyl acylation analyzed by primer extension （SHAPE）. The affinity strength and force properties between ribox02 and NAD^＋/ NADH were further detected by isothermal titratio calorimetry （ITC）. Finally, we accurately located the specific binding sites of NAD^＋/NADH with ribox02 RNA in the UV crosslinking. Results The secondary structure information of ribox02 was revealed and we found that the van der Waals force and hydrogen bonding is the interaction force between ribozyme ribox02 and NAD^＋/NADH,and the interaction force between ribox02 and NADH is more intense than it between ribox02 and NAD^＋;and the UV crosslinking experiment results showed that the specific binding sites of ribox02 and NAD^＋/NADH were 23G, 24U, 25U,38U, 72U, 73U, 74U,75G and 95G. Conclusions The secondary structure of ribox02 and its interaction with NAD^＋/ NADH reveals the affinity and specific binding sites between ribozyme ribox02 and NAD^＋/NADH.
作者
李丹彬
张静
陈东戎
LI Dan bin;ZHANG Jing;CHEN Dong tong(RNA Biology Laboratory, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China)
出处
《复旦学报（医学版）》
CAS
CSCD
北大核心
2018年第5期664-670,共7页
Fudan University Journal of Medical Sciences
基金
国家自然科学基金面上项目(31370107)
国家自然科学基金青年项目(31400050)
