Geoffrey Wickham

Rapid Determination of Sequence Selectivity and Stability of Alkylated Oligonucleotide Adducts by Electrospray Tandem Mass Spectrometry

The binding of the antitumor antibiotics, duocarmycin C2 (pyrindamycin A), duocarmycin C1 (pyrindamycin B), hedamycin, and DC92-B to self complementary oligonucleotides (ranging from 6 to 14-mers) has been studied using electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). The duocarmycins bind via non-covalent interactions in the minor groove of DNA with subsequent alkylation of the N3 atom of adenine. Hedamycin and DC92-B are intercalating, alkylating agents that target the N7 of guanines within 5′-CGT, and to a lesser extent, 5′-CGG sequences. We show here that the site(s) of alkylation by these ligands are strongly influenced by the location of high affinity binding sites within these short oligonucleotides. These data clearly demonstrate value of using ESI-MS/MS to pre-screen ligand–oligonucleotide complexes prior to performing more detailed structural studies, since subtle selectivity differences have been detected by this technique that were not evident from conventional sequencing studies on larger segments of DNA.

The observation of a hedamycin-d(CACGTG)2 covalent adduct by electrospray mass spectrometry

Covalent binding of the antitumour antibiotic hedamycin to the self‐complementary hexadeoxyribonucleotide 5′‐CACGTG‐3′ has been investigated by electrospray ionization mass spectrometry (ESI‐MS). Ions due to double‐stranded forms of the free 5′‐CACGTG‐3′ and the hedamycin‐5′‐CACGTG‐3′ adduct have been observed in ESI mass spectra and their identity has been confirmed by resolution of individual charge states in ESI‐MS spectra. Clear evidence that specific base‐paired associations are being observed in ESI‐MS is provided by the results of a titration experiment involving alkylated and non‐alkylated complementary strands. This work demonstrates the potential of this powerful new tool for studying ligand‐DNA binding.