无机离子对磷硫修饰DNA的碘催化断裂反应的显著影响  

The Significant Effect of Inorganic Ions on the Reaction of Iodine-Catalyzed Cleavage of Phosphorothioate DNA

作　　者：胡坤灵 汤若冰 安然[1,2] 梁兴国 Hu Kunling;Tang Ruobing;An Ran;Liang Xingguo(College of Food Science and Engineering,Ocean University of China,Qingdao 266003,China;Functional Laboratory of Marine Drugs and Biological Products,Pilot National Laboratory for Marine Science and Technology(Qingdao),Qingdao 266237,China)

机构地区：[1]中国海洋大学食品科学与工程学院,山东青岛266003 [2]青岛海洋科学与技术试点国家实验室海洋药物与生物制品功能实验室,山东青岛266237

出　　处：《中国海洋大学学报（自然科学版）》2022年第10期68-75,共8页Periodical of Ocean University of China

基　　金：国家重点研究发展计划项目(2019YFC1604603);山东省自然科学基金项目(ZR2019BC096)资助。

摘　　要：磷硫修饰DNA(PT-DNA)的断裂反应常被用于检测微生物基因组中PT-DNA的含量,但其检测准确性有待提高。本文研究了Tris-HCl、HEPES、磷酸盐等缓冲溶液和阳离子的种类、浓度对碘断裂PT-DNA的影响,提出了Tris分子参与断裂反应的机制。研究发现,Tris分子能使单链PT-DNA的断裂收率从5%提升至70%以上。对于单链PT-DNA断裂,Na^(+)、Mg^(2+)和Ca^(2+)等阳离子有显著的抑制作用;而双链PT-DNA断裂基本不受上述无机阳离子的影响。当用磷酸钠缓冲溶液代替Tris-HCl时,在低盐条件下PT-DNA只能产生微弱断裂,在高盐条件下双链状态PT-DNA的断裂反而获得了显著的提升;而改用HEPES缓冲液时,无论在高盐还是低盐条件下,PT-DNA的断裂效率都很低。研究结果表明,只有Tris分子结合了PT-DNA氧化后形成的亚磺酸基团才能实现高效断裂,而阳离子可通过调节Tris分子同亚磺酸基团的结合来影响断裂收率。PT-DNA的生物学功能可能是防止弱氧化剂对DNA的破坏(包括断裂或变异),并通过氧化应激启动抗氧化机制。本研究结果还表明,核酸药物在体内可能经氧化途径代谢或脱硫后被核酸酶分解,开发磷硫修饰的核酸类药物时需要考虑这一药物的代谢因素。The cleavage of phosphorothioate DNA(PT-DNA) is often used to detect PT-DNA in the microbial genome,but detection accuracy needs to be improved.In this study,we explored the effects of different buffers(Tris-HCl,HEPES,and phosphate) and the type and concentration of cations on the cleavage of PT-DNA.We proposed the mechanism of how Tris participates in the cleavage reaction.The result showed that Tris improved the cleavage yield of PT-DNA from 5% to 70%.Surprisingly,Na^(+),Mg^(2+),Ca^(2+),and other cations significantly inhibited the cleavage of the single-stranded PT-DNA while the breakage of double-stranded PT-DNA was not affected by these inorganic cations.Unexpectedly,when Tris-HCl was replaced by phosphate,only weak cleavage occurred at low salt concentrations.Nevertheless,the high concentrations of salt ions can significantly promote the breakage of double-stranded PT-DNA.When HEPES was used as a buffer,the cleavage yield was low regardless of salt concentration.The results suggested that the efficient cleavage is achieved by Tris combining with the ionized sulfinic group,which is formed by the oxidation of PT-DNA.Moreover,cations can affect the cleavage yield by regulating the binding between Tris and sulfinic group.We believed that PT-DNA can prevent weak oxidants from damaging DNA(including cleavage or mutations) and respond to oxidation signals.Our findings also indicated that nucleic acid drugs can be metabolized by oxidation or decomposed by nucleases after desulfurization in vivo,which needs to be consi-dered during the development of sulfur-modified nucleic acid drugs.

关 键 词：磷硫修饰DNA 碘氧化 核酸药物 基因保护 阳离子 

分 类 号：Q819[生物学—生物工程]