Structural characterization of carcinogen‐modified oligodeoxynucleotide adducts using matrix‐assisted laser desorption/ionization mass spectrometry

Abstract

The aim of this study was to determine the chemical structure of in vitro 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-modified oligodeoxynucleotides (ODNs) by exonuclease digestion and matrix-assisted laser desorption/ionization mass spectrometry. A single-stranded 11-mer ODN, 5-d(CCATCGCTACC), was reacted with N-acetoxy-PhIP, resulting in the formation of one major and eight minor PhIP-ODN adducts. A 10 min treatment of the major and one minor PhIP-ODN adduct with a 3-exonuclease, bovine intestinal mucosa phosphodiesterase (BIMP), and a 5-exonuclease, bovine spleen phosphodiesterase, results in inhibition of the primary exonuclease activity at deoxyguanosine (dG) producing 5-d(CCATCG(PhIP)) and 5-d(G(PhIP)CTACC) product ions, respectively. Post-source decay (PSD) of these enzymatic end products identifies dG as the sole modification site in two 11-mer ODN-PhIP adducts. PSD of the minor PhIP-ODN adduct digestion end product, 5-d(CCATCG(PhIP)), also reveals that the PhIP adducted guanine moiety is in an oxidized form. Prolonged treatment of the PhIP-ODN adducts at 37 • C with BIMP induces a non-specific, or endonuclease, enzymatic activity culminating in the formation of deoxyguanosine 5-monophosphate-PhIP (5-dGMP-PhIP). The PSD fragmentation pattern of the 5-dGMP-PhIP [M + H] + ion of the major adduct confirms PhIP binds to the C-8 position of dG. For the minor adduct, PSD results suggest that PhIP binds to the C-8 position of an oxidized guanine, supporting the hypothesis that this adduct arises from oxidative degradation, resulting in a spirobisguanidino structure.

Comparison of the nucleic acid covalent binding capacity of two nitro-substituted benzazolo[3,2-a]quinolinium salts upon enzymatic reduction

“…Evidence of the formation of an 2'-dG-NHBQ-38 adduct was confirmed by the detection of the molecular ion m/z 454.8, resulting from the [M+1] molecular ion m/z 528.3 (Figure 7). Higher levels of adduct formation would have allowed us to better define the structure of this [M+1] 528.3 molecular species; however, the loss of part of the ribose group, which is common under these ESI conditions (Brown, 2003) is a possible explanation.…”

“…A collision-induced dissociation experiment (CID of 528→454) revealed the origin of the m/z 454.8 signal, indicating the [M +1] + molecular ion m/z 528.2 as its precursor. Although the exact structure of this 454.8 molecular ion is not totally understood, it could represent the loss of part of the ribose group, which is common under ESI conditions (Brown, 2003;B. Van den Driessche et al, 2003;Rindgen, D., et al, 1995).…”

Sequence verification of oligonucleotides containing multiple arylamine modifications by enzymatic digestion and liquid chromatography mass spectrometry (LC/MS)

“…We identified two factors in this study that appear to enhance exonuclease activity relative to endonuclease activity. One was the lower temperature of the digestion, that being 24°C relative to 37°C as described in other investigations [9,10,13]. The second factor was a lower enzyme to substrate ratio in the digestion mixture.…”

Fluorescence Probing of Aminofluorene-Induced Conformational Heterogeneity in DNA Duplexes

“…The ODNs were sequenced using exonuclease (3′-5′ or 5′-3′) strategies described previously (23,24). Typically, 10 μg of each ODN was combined with ca.…”