Electrospray ionisation mass spectrometry (ESI-MS) holds considerable promise as a tool for probing the interactions of antitumour agents with DNA [1,2]. We have used ESI-MS to examine the covalent binding of a range of antitumour agents with self-complementary oligonucleotides as models for DNA. The observation of duplex forms of ligand-oligonucleotide adducts in ESI mass spectra has been found to correlate with the stability of these adducts in solution. For example, the antitumour antibiotic hedamycin, which both intercalates into and alkylates DNA, enhances the intensity of duplex ions in ESI mass spectra. In contrast, cisplatin destabilises the duplex forms both in solution and the gas phase.
Determination of both the extent of binding and the site of binding of a ligand to oligonucleotides of different sequences is important for understanding the mode of action of the ligand. We have found that ESI combined with tandem mass spectrometry (MS-MS) has the potential to rapidly establish to which base(s) on a sequence the ligand is bound. ESI-MS-MS spectra of free oligonucleotides are dominated by fragments arising from non-sequence dependent losses of the bases from the oligonucleotide backbone. In contrast, ESI-MS-MS spectra of the [M-2H]2- ion of the covalent adduct formed between hedamycin and 5'-CACGTG-3', for example, shows cleavages directed by the position of the ligand, which are thus indicative of the site of binding, in this case to the guanine in the sequence 5' CGT. This is consistent with NMR and sequencing studies of hedamycin.
The extent of binding of cisplatin to the self-complementary oligonucleotides 5'-AGGCCT-3', 5'-CACGTG-3' and 5'-TACGTA-3' has been determined by both ESI-MS and HPLC. There is good correlation between the results of the two techniques, with in both cases, the greatest degree of binding observed for 5'-AGGCCT-3' which incorporates a preferred GG binding site for cisplatin.
This study demonstrates the considerable potential of ESI and ESI-MS-MS for the characterisation of ligand-DNA adducts which in turn should aid in the development of improved antitumour agents.