Related Experiment Videos
Mass spectrometric sequencing of site-specific carcinogen-modified oligodeoxyribonucleotides containing bulky
1American Health Foundation, Valhalla, New York 10595, USA.
Abstract:
Site-specific carcinogen-modified oligonucleotides are often used in site-directed mutagenesis and other biological and biochemical studies of structure-function relationships. Postsynthetic analysis and confirmation of the sites of carcinogen binding in such oligonucleotides is an important step in the characterization of these site-specific carcinogen-DNA adducts. It is shown here that negative ion mode electrospray tandem mass spectrometry methods and collision-induced dissociation offer a rapid and convenient approach for the sequencing of products derived from the reaction of the carcinogenic and mutagenic metabolite of benzo[a]pyrene, the diol epoxide r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), with the 11-mer oligonucleotide d(CATGCGGCCTAC). The site of reaction of anti-BPDE with either one of the three dG residues in this oligonucleotide can be accurately established by comparing the mass/charge ratios of the observed collision-induced dissociation fragments with calculated values.
Insights
Researchers used mass spectrometry to identify where the carcinogen benzo[a]pyrene binds to DNA. This method rapidly sequences carcinogen-DNA adducts, confirming binding sites for biological studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Site-specific carcinogen-modified oligonucleotides are crucial for studying DNA structure-function relationships.
- Characterizing carcinogen-DNA adducts requires confirming the exact sites of carcinogen binding post-synthesis.
Purpose of the Study:
- To demonstrate a rapid and convenient method for sequencing carcinogen-DNA adducts.
- To accurately establish the binding site of benzo[a]pyrene diol epoxide (BPDE) on an oligonucleotide.
Main Methods:
- Utilizing negative ion mode electrospray tandem mass spectrometry.
- Employing collision-induced dissociation (CID) for fragment analysis.
- Comparing experimental CID fragment mass/charge ratios with calculated values.
Main Results:
- Successfully sequenced products from the reaction of anti-BPDE with the oligonucleotide d(CATGCGGCCTAC).
- Accurately identified the specific deoxyguanosine (dG) residue modified by anti-BPDE.
- Demonstrated the utility of mass spectrometry for characterizing carcinogen-DNA adducts.
Conclusions:
- Negative ion mode electrospray tandem mass spectrometry coupled with CID is a powerful tool for analyzing carcinogen-DNA adducts.
- This technique enables precise determination of carcinogen binding sites on oligonucleotides.
- The findings facilitate further research into the structure-function relationships of carcinogen-modified DNA.