Effects of Base Flipping on Guanine-Metal Electrode Transfer

这一无机化学、生物无机化学和金属有机化学计划和多学科活动办公室在GOALI活动下的联合奖项支持北卡罗来纳大学教堂山分校霍尔登·索普教授的实验室研究。这项工作是为了开发一种新的生物分析方法，通过结合DNA和RNA的蛋白质来检测碱基翻转。最初的研究将用已知的将碱基从核酸中取出的蛋白质来进行，这会使它们的互补碱基“孤立”地从核酸中挤出，从而使其暴露在光化学激发的(Pt2(POP)4)4-或(Ru(Bpy)3)3的氧化中，后者是通过电化学产生的。该方法是针对鸟嘌呤(G)残基的，该残基在氧化时产生8-O-G。在哌啶存在的情况下，8-O-G位被切割，因此核酸测序揭示了碱基(胞嘧啶)翻转的位置。M.Hhai目前正在研究将C转化为甲基化的蛋白质。Goali的合作涉及位于马萨诸塞州剑桥的新英格兰Biolabs(NEB)，该公司是分子生物学限制酶的主要供应商。北卡罗来纳大学的一名或多名学生将在NEB主持克隆和过度表达感兴趣的酶，并将这一专业知识传回北卡罗来纳大学。凝胶分析结果将转移到NEB。如果成功，果胶相互作用可能为研究通过碱基翻转处理核酸的蛋白质提供一个重要的新试剂。这项由Goali活动资助的研究探索了两种新的DNA裂解剂作为由几种重要的DNA结合蛋白进行碱基翻转的指示剂。

This joint award in the Inorganic, Bioinorganic and Organometallic Chemistry program and the Office of Multidisciplinary Activities under the Grant Opportunities for Academic Liaison with Industry (GOALI) activity supports research in the laboratory of Professor Holden Thorp at the University of North Carolina at Chapel Hill. The work is to develop a new bioanalytical method of detecting base flipping by proteins that bind DNA and RNA. Initial studies will be done with proteins known to turn bases out of the nucleic acid, which leaves their complementary base `orphaned` and so extruded from the nucleic acid thereby exposing it to oxidation by photochemically excited (Pt2(pop)4)4- or by (Ru(bpy)3)3+, which is generated electrochemically. The method is specific to guanine (G) residues, which upon oxidation yields 8-O-G. In the presence of piperidine, the 8-O-G site is cleaved, so that nucleic acid sequencing reveals the site of base (cytosine) flipping. M.HhaI is now under study, which turns C into the protein for methylation. The GOALI collaboration involves New England Biolabs (NEB) in Cambridge MA, which is a primary supplier for restriction enzymes for molecular biology. One or more students from UNC would be hosted at NEB to clone and overexpress enzymes of interest and to transfer this expertise back to UNC. Gel assay results would be transferred to NEB. If successful, the GOALI interaction could provide an important new reagent for the study of proteins that process nucleic acids through base flipping. This research funded under the GOALI activity explores the use of two new DNA cleaving agents as indicators of base flipping by several important DNA binding proteins.