A selective, noncovalent assay for base flipping in DNA.
Base flipping, the conformational change of a nucleobase to an extrahelical position, is a key step in the enzymatic repair of damaged DNA. An assay that can detect the flipped-out species in free solution without covalent modification of the DNA would be desirable. The design and synthesis of a simple, sensitive, and rapid assay using specific noncovalent binding to pyrimidines by zinc-cyclen and a commonly used fluorescent reporter group, dansyl, is reported. The binding of the zinc-cyclen unit to a flipped-out thymine base results in a change in the fluorescent properties of the dansyl group that is distinct from nonspecific binding to duplex DNA or intercalation into either the flipped-in or flipped-out species. The assay was tested using fluorescence spectroscopy and detection at 533 +/- 5 nm with normal and abasic duplex DNA as negative and positive controls. The data obtained are fitted to a one-site binding model to determine the equilibrium constant for the two-step process involving base flipping and binding to be approximately 10-6 M.
O'Neil
Lauren L
LL
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556-5670, USA.
Wiest
Olaf
O
D015075:2-Aminopurine / Q000737:chemistry; D000225:Adenine / Q000032:analysis / Q000737:chemistry; D020029:Base Pairing; D004247:DNA / Q000032:analysis / Q000737:chemistry*; D005453:Fluorescence; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D013941:Thymine / Q000032:analysis / Q000737:chemistry; D017186:Titrimetry
D015075:2-Aminopurine; D004247:DNA; D000225:Adenine; D013941:Thymine