Chemical consequences of the incorporation of 5-fluorouracil into DNA as studied by NMR

Abstract

The cytotoxic analogue of thymine, 5-fluorouracil (Uf), is known to be incorporated into DNA in biological systems. This abnormal base has been synthetically incorporated into short DNA oligomers. The ionization of the N-3 proton of this base within DNA oligomers was measured by observation of the 19F chemical shift at varying pH values. The pKa values for the Uf ring of dTpdUfpdT and dApdUfpdA were determined to be 7.84 and 7.9, respectively. The self-complementary 12-mers d(G-C-G-C-A-A-T-Uf-G-C-G-C) and d(C-G-A-T-Uf-A-T-A-A-T-C-G) were synthesized, and 1H NMR was used to compare the helix dynamics and stability of the interstrand imino proton hydrogen bonds with those of the 12-mers d(G-C-G-C-A-A-T-T-G-C-G-C) and d(C-G-A-T-T-A-T-A-A-T-C-G). The N-3 hydrogen bond of the A-Uf base pair was less stable than the corresponding hydrogen bond in A-T base pairs in the same helix, and the A-Uf base pair was less stable than the A-T base pair in the analogous position of the control helix. The observed temperature-dependent dynamics and NMR melting temperatures of the control and dUf-containing oligomers were similar.