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Probing ligand binding to duplex DNA using KMnO4 reactions and electrospray ionization tandem mass spectrometry.
- Source :
-
Analytical chemistry [Anal Chem] 2007 Jun 15; Vol. 79 (12), pp. 4636-47. Date of Electronic Publication: 2007 May 18. - Publication Year :
- 2007
-
Abstract
- An electrospray ionization tandem mass spectrometry (ESI-MS/MS) strategy employing the thymine-selective KMnO4 oxidation reaction to detect conformational changes and ligand binding sites in noncovalent DNA/drug complexes is reported. ESI-MS/MS is used to detect specific mass shifts of the DNA ions that are associated with the oxidation of thymines. This KMnO4 oxidation/ESI-MS/MS approach is an alternative to conventional gel-based oxidation methods and affords excellent sensitivity while eliminating the reliance on radiolabeled DNA. Comparison of single-strand versus duplex DNA indicates that the duplexes exhibit a significant resistance to the reaction, thus confirming that the oxidation process is favored for unwound or single-strand regions of DNA. DNA complexes containing different drugs including echinomycin, actinomycin-D, ethidium bromide, Hoechst 33342, and cis-C1 were subjected to the oxidation reaction. Echinomycin, a ligand with a bisintercalative binding mode, was found to induce the greatest KMnO4 reactivity, while Hoechst 33342, a minor groove binder, caused no increase in the oxidation of DNA. The oxidation of echinomycin/DNA complexes containing duplexes with different sequences and lengths was also assessed. Duplexes with thymines closer to the terminal ends of the duplex demonstrated a greater increase in the degree of oxidation than those with thymines in the middle of the sequence. Collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) experiments were used to determine the site of oxidation based on oligonucleotide fragmentation patterns.
- Subjects :
- Base Sequence
Benzimidazoles chemistry
Benzimidazoles metabolism
Binding Sites
DNA metabolism
Dactinomycin chemistry
Dactinomycin metabolism
Echinomycin chemistry
Echinomycin metabolism
Ethidium chemistry
Ethidium metabolism
Ligands
Oligonucleotides chemistry
Oligonucleotides metabolism
Oxidation-Reduction
Pharmaceutical Preparations chemistry
Thymine chemistry
Thymine metabolism
DNA chemistry
Pharmaceutical Preparations metabolism
Potassium Permanganate chemistry
Spectrometry, Mass, Electrospray Ionization
Subjects
Details
- Language :
- English
- ISSN :
- 0003-2700
- Volume :
- 79
- Issue :
- 12
- Database :
- MEDLINE
- Journal :
- Analytical chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 17508717
- Full Text :
- https://doi.org/10.1021/ac070145p