A versatile platform for single-molecule enzymology of restriction endonuclease  

作　　者：Xin Wang Jingyuan Nie Yi Li Hai Pan Peng Zheng Meng Qin Yi Cao Wei Wang 

机构地区：[1]National Laboratory of Solid State Microstructure School of Physics,Nanjing University 22 Hankou Road Nanjing,Jiangsu 210093,P.R.China [2]School of Chemistry and Chemical Engineering Nanjing University 163 Xianlin Avenue Nanjing,Jiangsu 210023,P.R.China

出　　处：《Journal of Innovative Optical Health Sciences》2019年第1期29-38,共10页创新光学健康科学杂志（英文）

基　　金：The authors greatly appreciate the-nancial support from National Natural Science Foundation of China(Grant Nos.21522402,11674153,11374148,11334004 and 21771103);the Fundamental Research Funds for the Central Universities(Nos.020414380070,020414380050 and 020414380058);Natural Science Foundation of Jiangsu Province(No.BK20160639)and the Shuangchuang Program of Jiangsu Province.

摘　　要：Enzymes are the major players for many biological processes.Fundamental studies of the enzymatic activity at the single-molecule level provides important information that is otherwise inaccessible at the ensemble level.Yet,these single-molecule experiments are technically di±cult and generally require complicated experimental design.Here,we develop a Holliday junction(HJ)-based platform to study the activity of restriction endonucleases at the single-molecule level using single-molecule FRET(sm-FRET).We show that the intrinsic dynamics of HJ can be used as the reporter for both the enzyme-binding and the substrate-release events.Thanks to the multiple-arms structure of HJ,the fluorophore-labeled arms can be different from the surface anchoring arm and the substrate arm.Therefore,it is possible to independently change the substrate arm to study different enzymes with similar functions.Such a design is extremely useful for the systematic study of enzymes from the same family or enzymes bearing different pathologic mutations.Moreover,this method can be easily extended to study other types of DNA-binding enzymes without too much modi fication of the design.We anticipate it can find broad applications in single-molecule enzymology.

关 键 词：Single molecule Forster resonance energy transfer ENZYMOLOGY KINETICS holliday junction 

分 类 号：R318[医药卫生—生物医学工程]