We demonstrate herein that hemin-graphene hybrid nanosheets(H-GNs)can differentiate hemin-binding DNA aptamers(i.e.,guanine-rich DNA sequences that form G-quadruplex structures upon binding hemin)and nonspecific singl...We demonstrate herein that hemin-graphene hybrid nanosheets(H-GNs)can differentiate hemin-binding DNA aptamers(i.e.,guanine-rich DNA sequences that form G-quadruplex structures upon binding hemin)and nonspecific single-stranded oligonucleotides colorimetrically.By exploiting the tendency of the H-GNS to aggregate under high ionic strength and their inherent catalytic activities,excess H-GNS were removed and the amount of H-GNs remaining in the supernatant was quantified.Subsequently,the binding affinity of hemin-binding DNA aptamers to the H-GNS was determined upon titration with the DNA.With the conformity of the experimental data to the Langmuir adsorption isotherm,hemin is hypothesized to be sandwiched between the G-quadruplex DNA and graphene forming a ternary complex as a result of the specific bind-ing of the aptamer with the H-GNS.The binding affinity of the aptamer is not as strong as that of hemin/G-quadruplex in solution,which is attributed to electronic effects imparted by the graphene surface.The catalytic activity of the resulting complex,however,was confirmed to be higher than that of H-GNs alone.展开更多
文摘【目的】利用大肠杆菌表达纯化嗜热脱铁去硫弧菌(Deferribacter desulfuricans)解旋酶DePif1,并对其结合与解旋DNA的活性进行分析,为Pif1家族解旋酶结构和功能的阐明奠定基础。【方法】将促溶标签SUMO编码序列和人工合成的DePif1解旋酶编码序列依次连入pET15b载体,获得重组融合表达载体pET15b-SUMO-DePif1,然后将其导入E.coli BL21(DE3)菌株进行诱导表达;利用Ni-NTA亲和层析柱获得融合蛋白,SUMO蛋白酶酶切去除融合标签,再经Heparin和Ni-NTA柱分离获得无标签的纯化重组DePif1蛋白;采用荧光各向异性分析,研究pH和NaCl浓度对DePif1与DNA结合的影响及DePif1与不同底物(单链DNA、双链DNA和G4-DNA)的结合特性;使用基于荧光共振能量转移的stopped-flow技术,分析DePif1对不同底物(G4-DNA with 5′26nt tail和dsDNA with 5′26nt tail)的解旋活性。【结果】每升菌液可获得9 mg纯度大于95%的DePif1解旋酶。DePif1结合不同DNA底物的强度依次为G4-DNA>单链DNA>双链DNA,其对G4-DNA的解旋活力大于双链DNA。【结论】成功表达并纯化了嗜热脱铁去硫弧菌Pif1解旋酶,并证明其具有特异的G4-DNA结合和解旋能力。
基金the financial supports of Natural Sciences and Engineering Research Council(NSERC)of Canada and National Natural Science Foundation of China(no.21775095)。
文摘We demonstrate herein that hemin-graphene hybrid nanosheets(H-GNs)can differentiate hemin-binding DNA aptamers(i.e.,guanine-rich DNA sequences that form G-quadruplex structures upon binding hemin)and nonspecific single-stranded oligonucleotides colorimetrically.By exploiting the tendency of the H-GNS to aggregate under high ionic strength and their inherent catalytic activities,excess H-GNS were removed and the amount of H-GNs remaining in the supernatant was quantified.Subsequently,the binding affinity of hemin-binding DNA aptamers to the H-GNS was determined upon titration with the DNA.With the conformity of the experimental data to the Langmuir adsorption isotherm,hemin is hypothesized to be sandwiched between the G-quadruplex DNA and graphene forming a ternary complex as a result of the specific bind-ing of the aptamer with the H-GNS.The binding affinity of the aptamer is not as strong as that of hemin/G-quadruplex in solution,which is attributed to electronic effects imparted by the graphene surface.The catalytic activity of the resulting complex,however,was confirmed to be higher than that of H-GNs alone.
