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

Abstract

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.