DNA硫修饰结合蛋白识别序列的表征与拓展促进新型核酸检测技术发展  

Profile and relaxation of sequence-specificity of DNA sulfur binding domains facilitate new nucleic acid detection platform

作　　者：帅玉婷 许安安 李甲乙 韩照晰 马蒂尼 段海容 王馨叶 蒋岚 张敬宇 谭高翼 刘雪婷 赵一雷 童垚俊 王申林 贺新义 邓子新 刘光 张立新 Yuting Shuai;Anan Xu;Jiayi Li;Zhaoxi Han;Dini Ma;Hairong Duan;Xinye Wang;Lan Jiang;Jingyu Zhang;Gao-Yi Tan;Xueting Liu;Yi-Lei Zhao;Yaojun Tong;Shenlin Wang;Xinyi He;Zixin Deng;Guang Liu;Lixin Zhang(State Key Laboratory of Bioreactor Engineering,School of Biotechnology,East China University of Science and Technology(ECUST),Shanghai 200237,China;State Key Laboratory of Microbial Metabolism,Joint International Research Laboratory of Metabolic&Developmental Sciences,School of Life Sciences&Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]State Key Laboratory of Bioreactor Engineering,School of Biotechnology,East China University of Science and Technology(ECUST),Shanghai 200237,China [2]State Key Laboratory of Microbial Metabolism,Joint International Research Laboratory of Metabolic&Developmental Sciences,School of Life Sciences&Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China

出　　处：《Science Bulletin》2023年第16期1752-1756,M0003,共6页科学通报（英文版）

基　　金：supported by the National Key Research and Development Program of China(2020YFA0907800,2022YFC3400200,and 2022YFA0912200);the National Natural Science Foundation of China(31900060);the Shanghai Pilot Program for Basic Research-Shanghai Jiao Tong University(21TQ1400204);the Natural Science Foundation of Shanghai(20ZR1414500)。

摘　　要：DNA硫修饰是一种DNA磷酸骨架上的非桥接氧原子被硫原子所取代的修饰,属于原核生物表观遗传标志物之一,它对细菌防御系统、抗氧化应激和基因调控等生命过程具有重要影响.DNA硫修饰可以被硫修饰依赖限制性核酸内切酶的硫结合域SBD特异性识别,这使其具有被开发为新型生物技术工具的潜力.然而目前关于SBD蛋白识别序列的研究仍比较粗浅且仅限于少数天然序列模式,这极大限制了其应用开发.因此,我们建立了一种针对SBD蛋白识别序列表征的技术(SBS-seq),用于对SBD的完整识别序列进行高分辨率表征.而且,采用在DNA上串联多个硫修饰的方案来提高SBD的结合亲和力并拓展了其序列识别范围.在此基础上,我们进一步优化了基于SBD和硫修饰DNA的新型核酸检测技术.本工作为硫修饰依赖限制性核酸内切酶的研究提供了思路和范例,并促进了硫修饰DNA相关生物技术的应用.DNA phosphorothioate(PT)-modification,with a non-bridging oxygen in the phosphodiester backbone substituted by sulfur,is an epigenetic marker in prokaryotes and involved in the bacterial defense system,anti-oxidative stress,and gene regulation[1–4].PT-modification could be specifically recognized by sulfur binding domains(SBD)of PT-dependent restriction endonucleases(REases)[5,6],making it a potential tool for enabling biotechnology development[7].However,the unclear recognition sequence-range of SBDs limits the application.Here,we report a technique named sulfur binding specificity-sequencing(SBS-seq)for highresolution characterization of the complete sequence-range of SBDs.We then employed tandem PT-modifications to improve the binding affinity and extend the sequence-range.The strong affinity and broad range facilitate a new nucleic acid detection platform based on SBD and PT-DNA.Our work provides insights for research on modification-dependent REases and facilitates biotechnology applications of PT-DNA.

关 键 词：核酸检测技术 限制性核酸内切酶 SBD 序列模式 识别序列 防御系统 硫原子 抗氧化应激 

分 类 号：Q503[生物学—生物化学]