凝胶原位探测c-Myc G-四链体的结构多态性  

In-Gel Probing Polymorphic Structures of G-Quadruplexes Derived From c-Myc Promoter

作　　者：常天俊[1] 武俐[1] 卞文秀 邴涛[2] 赵同谦[1] 上官棣华[2] CHANG Tian-Jun;WU Li;BIAN Wen-Xiu;BING Tao;ZHAO Tong-Qian;SHANGGUAN Di-Hua(Department of Biology,Institute of Resources and Environment,Henan Polytechnic University,Jiaozuo 454000,China;Beijing National Laboratory for Molecular Sciences,Key Laboratory of Analytical Chemistry for Living Biosystems,Institute of Chemistry,Chinese Academy of Sciences,Beijing 100190,China)

机构地区：[1]河南理工大学资源环境学院,焦作454000 [2]中国科学院化学研究所,北京分子科学国家实验室,活体分析化学院重点实验室,北京100190

出　　处：《分析化学》2020年第3期323-331,共9页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.U1704241);中原科技创新领军人才项目(No.194200510010);河南省自然科学基金项目(No.162300410111)资助~~

摘　　要：G-四链体(G4)在某些条件下可形成聚集结构,丰富了其结构多态性,但影响了其结构与功能研究。本研究建立了对平行G4 c-Myc及衍生序列形成的分子内结构和分子间聚集体同时分离与原位探测的方法,并讨论了末端碱基对c-Myc形成聚集体的影响。首先通过荧光光谱研究了硫磺素T(Thioflavin T,ThT)与G4的相互作用,发现ThT对含不同A/T末端的c-Myc响应不同;比较典型分子间平行以及反平行单体G4,发现ThT结合分子间平行G4能使其荧光强烈激活。用ThT对凝胶中的G4条带特异性染色,再用Stains-all对所有DNA条带染色,通过ImageJ获得DNA条带经双重染色的光密度比值,发现分子间平行单体G4或分子间G4聚集体的光密度比值大于1,分子内平行G4的光密度比值约为0.5,非G4结构的光密度比值接近0;发现末端碱基影响c-Myc聚集体的形成,在K+溶液中增加末端碱基利于形成G4聚集体,其中T作用更强;在Na+溶液中只有末端含两个或以上的A利于其形成G4聚集体。本研究利用特异性与非特异性的染料组合在胶上原位探测G4构型多态性的策略,将有助于G4多态性及其构-效关系的研究。Some G-quadruplex(G4)forming sequences are able to form multimers under physiological conditions,which limits the structure-activity study of G4s.Here we reported an in-gel visual method to probe the conformational polymorphism of the derivatives of a parallel G4-forming sequence,c-Myc by flanking with different A/T bases and typical intermolecular G4-forming sequences.Fluorescence and circular dichroism(CD)spectra showed that the interaction of c-Myc derivatives with Thioflavin T(ThT)caused different fluorescence enhancements of ThT,and intermolecular parallel G4s caused higher fluorescence enhancement of ThT than that of intramolecular parallel and intermolecular antiparallel G4s.These DNA sequences were separated on native gels and stained by ThT for G4 and then by Stains-all(dye)for all DNA structures.Most of the sequences adopted parallel G4 multimers and intramolecular G4s in the presence of K+,meanwhile only a part of them formed G4 multimers and intramolecular structures in the presence of Na+.The optical density(OD)ratio of DNA bands stained by ThT and Stains-all were derived by ImageJ,and the ratios of G4 multimers were higher than 1.0,intramolecular parallel G4s were about 0.5,and non-G4s were near 0.Furthermore,the flanking nucleotides were favorable to the formation of multimers of c-Myc in K+solutions,especially those flanking with T;while in Na+solutions,only those sequences flanking with two or more A preferred to form G4 multimers.The results suggested the current strategy had the potential for the polymorphic study of G4s.

关 键 词：G-四链体 原位探测 结构多态性 分子识别 荧光探针 

分 类 号：O657.3[理学—分析化学] O641[理学—化学]