DHPLC and MS studies of a photoinduced intrastrand cross-link in DNA labeled with 5-bromo-2′-deoxyuridine

• We study the photodegradation of an oligonucleotide sensitized by 5-bromouracil.
• Completely denaturing HPLC (DHPLC) is a suitable method for DNA damage assays.
• Covalent intrastrand dimer between adenine and uracil is the only photoproduct.
• Photodissociation of the C5-Br bond rather than electron transfer leads to the dimer.

It is well known that the replacement of thymidine with 5-bromo-2′-deoxyuridine (BrdU) in DNA sensitizes it to UVB light. Irradiation of a biopolymer substituted in such a way leads to manifold kinds of DNA damage, such as intrastrand cross-links, single- and double-strand breaks or alkali-labile sites that were studied in the past with a broad spectrum of analytical methods. Here, we demonstrate that completely denaturing high-performance liquid chromatography (DHPLC), underestimated so far in DNA damage studies, could act as an inexpensive, and high-resolution substitute for the commonly employed gel electrophoresis. We report on the DHPLC/mass spectrometry (MS) analyses of photolytes obtained with the UV irradiation of aqueous solutions containing 40 base pairs of a long, double-stranded oligonucleotide labeled with BrdU in one of its strands. The UV-product was detected by HPLC at a temperature of 70 °C. Subsequent MS analysis with electrospray ionization (ESI-MS) of the photolyte, enzymatic digestion of the irradiated material and HPLC and MS analysis (LC–MS) of the digest demonstrated unequivocally that an intrastrand covalent dimer, involving adenine and uracil, is formed in the irradiated system.

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