Effects of Base Flipping on Guanine-Metal Electrode Transfer

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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.