2-Amino purine as a probe of DNA deformability in damaged DNA.

Abstract

2-Amino purine (2AP) is an isomer of adenine (6-aminopurine) that, unlike the canonical DNA bases, is highly fluorescent when free in solution. The fluorescence quantum yield of 2AP in water is 0.68, and its fluorescence intensity decay is monoexponential with a single lifetime of 10-11 ns. When incorporated into DNA, 2AP fluorescence is quenched strongly by interactions with its neighboring bases. Inter-base interactions in duplex DNA give rise to a multiexponential decay that reflects the highly heterogeneous environment sensed by the probe. We used steady-state and time-resolved 2AP fluorescence to probe DNA dynamics around a lesion. In this work, we focused on uracil, a highly mutagenic and common lesion in DNA. In the cell, uracil is removed by Uracil DNA-glycosylase (UNG). UNG excision efficiency depends on DNA sequence, and this research is part of a broader study aimed to elucidate the underlying principles that dictate UNG substrate specificity. Duplex DNA substrates were prepared by annealing uracil-containing oligonucleotides with complementary strands containing 2AP opposite to the uracil. Fluorescence lifetimes were measured with 40 ps resolution, and results were analyzed together with the measured fluorescence quantum yields to calculate the fraction of ultra-quenched 2AP molecules. A higher fraction indicates a higher fraction of highly stacked (and therefore efficiently quenched) 2AP molecules, which we interpret as evidence for a less deformable substrate. This interpretation is supported by NMR imino proton exchange measurements and molecular dynamics simulations on the same substrates.