金属离子对二甲亚砜依赖的RNA切割型脱氧核酶催化活性的影响  

Effect of Metal Ions on the Catalytical Activity of a DMSO-dependent RNA-cleaving DNAzyme

作　　者：崔力 李素慧 郑星 常天俊[1] 邴涛 CUI Li;LI Suhui;ZHENG Xing;CHANG Tianjun;BING Tao(Institute of Resources and Environment,Henan Polytechnic University,Jiaozuo 454003,China;Hangzhou Institute of Medicine(HIM),Chinese Academy of Sciences,Hangzhou 310022,China)

机构地区：[1]河南理工大学资源环境学院,焦作454003 [2]中国科学院杭州医学研究所,杭州310022

出　　处：《高等学校化学学报》2024年第4期35-42,共8页Chemical Journal of Chinese Universities

基　　金：国家自然科学基金(批准号:U1704241,22274139);浙江省自然科学基金(批准号:YXD24B0401);浙江省“尖兵”“领雁”研发攻关计划(批准号:2023SDYXS0002);国家卫生健康委科学研究基金-浙江省卫生健康重大科技计划(批准号:WKJ-ZJ-2424);浙江省核酸适体与临床诊治重点实验室开放基金项目(批准号:2022ASC001)资助。

摘　　要：在高浓度有机溶剂中工作的RNA切割型脱氧核酶(RNA-cleaving DNAzyme,RCD)及其构筑的分子器件不仅拓展了DNA作为酶的能力,还可将功能核酸推进新的应用领域.本文研究了一个需要二甲亚砜才能工作的RCD(命名为E3)对金属离子的需求,发现二价金属离子对其催化活性至关重要,活性顺序为Zn^(2+),Mg^(2+)>Fe^(2+)>Pb^(2+)>Mn^(2+)>Co^(2+).以Mg^(2+)或Zn^(2+)为辅因子,表征了E3的速率-pH值关系及其与二者的结合比例.E3的催化速率-pH曲线在Mg^(2+)存在下为“钟形”,高速率的pH值范围为7.0~9.0;Zn^(2+)存在下为“尖峰”,速率最高时pH=7.0;E3与Mg^(2+)和Zn^(2+)的数量结合比例均为1∶1.另外,E3以Fe^(2+)为辅因子时易失活,Fe^(2+)被氧化成Fe^(3+)是失活的关键,加入还原剂可使其复活.进一步研究发现,Cu^(2+),Fe^(3+)和Ni^(2+)等金属离子可抑制Mg^(2+)或Zn^(2+)的作用,使E3的催化活性急剧降低.本文研究结果为理解E3的性质及催化机制提供了有用信息.Engineering RNA-cleaving DNAzymes(RCDs)and RCD-based molecular devices that are highly functional in high content of organic co-solvents may not only expand the ability of DNAs as enzymes,but also promote functional nucleic acids for novel applications.In this work,we have investigated the requirement of divalent metal ions for a dimethyl sulfoxide(DMSO)-dependent RCD(named E3)that we previously reported.The divalent metal ions were proved to be crucial for E3 to function in 35%(volume fraction)DMSO,and the metal ions with the ability to activate E3 followed the order:Zn^(2+),Mg^(2+)>Fe^(2+)>Pb^(2+)>Mn^(2+)>Co^(2+).The rate-pH profile of E3 in Mg^(2+)showed a“bell”shaped curve with a plateau at the pH range from 7.0 to 9.0,yet a sharp peak at pH value of 7.0 in Zn^(2+).Moreover,both Zn^(2+)and Mg^(2+)bound to E3 at a number ratio of 1∶1.We also observed that E3 could be activated by Fe^(2+)in 35%DMSO,but quickly inactive by the rapid oxidation of Fe^(2+)to Fe3+.This inactivation of E3 could be further rescued by the addition of ascorbic acid.In addition,the catalytic activity of E3 in the presence of Zn^(2+)or Mg^(2+)was sharply inhibited with the competition of Cu^(2+),Ni^(2+),and Fe3+.These results are helpful for understanding the characterization and catalytical mechanism of the DMSO-dependent RCD.

关 键 词：RNA切割型脱氧核酶 功能核酸 有机溶剂 金属离子 

分 类 号：O629.8[理学—有机化学]