ABSTRACT
The reactions of two well-known chemical probes, glyoxal and potassium permanganate (KMnO(4)), with oligodeoxynucleotides were monitored by electrospray ionization (ESI) mass spectrometry to evaluate the influence of the sequence of DNA, its secondary structure, and interactions with associated ligands on the reactivity of the two probes. Glyoxal, a guanine-reactive probe, incorporated a mass shift of 58 Da, and potassium permanganate (KMnO(4)) is a thymine-reactive probe that resulted in a mass shift of 34 Da. The reactions depended on the accessibility of the nucleobases, and the peak abundances of the adducts in the ESI-mass spectra were used to quantify the extent of the chemical probe reactions. In this study, both mixed-base sequences were studied as well as control sequences in which one reactive site was located at the terminus or center of the oligodeoxynucleotide while the surrounding bases were a second, different nucleobase. In addition, the reactions of the chemical probes with non-covalent complexes formed between DNA and either actinomycin D or ethidium bromide, both known to interact with single strand DNA, were evaluated.
ABSTRACT
Infrared multiphoton dissociation (IRMPD) of thymine-rich oligodeoxynucleotides in a linear ion-trap mass spectrometer affords far more extensive fragmentation than conventional collision-induced dissociation (CID). For oligodeoxynucleotides containing one non-thymine base, CID results primarily in cleavage on the 3' side of the non-thymine nucleobase, whereas IRMPD results in cleavages between all the nucleobases and thus provides complete sequence coverage. Furthermore, for oligodeoxynucleotides containing a single non-thymine base, it is shown that the full series of diagnostic sequence ions observed in the IRMPD mass spectra arise from secondary dissociation of the two primary products formed from the initial cleavage site located next to the non-thymine base.
