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Synthesis of a duplex oligonucleotide containing a nitrogen mustard interstrand DNA-DNA cross-link
J O Ojwang1, D A Grueneberg, E L Loechler
1Department of Biology, Boston University, Massachusetts 02215.
Abstract:
Many cancer chemotherapeutic agents react with DNA and give adducts that block DNA replication, which is thought to result in cytotoxicity, especially in rapidly proliferating cells such as cancer cells. One class of these agents is bifunctionally reactive (e.g., the nitrogen mustards) and forms DNA-DNA cross-links. It is unknown whether inter- or intrastrand cross-links are more effective at blocking DNA replication. To evaluate this, a DNA shuttle vector is being constructed with an interstrand cross-link at a unique site. In the first step of this project, a duplex oligonucleotide containing an interstrand cross-link is isolated by denaturing polyacrylamide gel electrophoresis from the reaction of nitrogen mustard with two partially complementary oligodeoxynucleotides. The purified oligonucleotide product is characterized and shown to be cross-linked in a 5'-GAC-3' 3'-CTG-5' sequence by a nitrogen mustard moiety that is bound at the N(7)-position of the guanines in the opposing strands; the glycosylic bonds of these guanine adducts are stabilized in their corresponding imidazole ring-opened form. Nitrogen mustard is shown to react with a variety of oligonucleotides and, based upon these results, its preferred targets for interstrand cross-linking are 5'-GXC-3' sequences, where X can be any of the four deoxyribonucleotide bases.
Insights
Nitrogen mustards create DNA interstrand cross-links, which are crucial for cancer chemotherapy. This study identifies preferred DNA sequences for these cross-links, aiding in the development of more effective cancer treatments.
Area of Science:
- Molecular Biology
- Cancer Research
- Medicinal Chemistry
Background:
- Cancer chemotherapeutic agents often function by damaging DNA, leading to cytotoxicity in rapidly dividing cancer cells.
- Bifunctional agents like nitrogen mustards form DNA-DNA cross-links, but the relative efficacy of interstrand versus intrastrand cross-links in blocking DNA replication is unclear.
Purpose of the Study:
- To construct a DNA shuttle vector containing a site-specific interstrand cross-link.
- To investigate the DNA sequence preferences of nitrogen mustards for interstrand cross-linking.
Main Methods:
- Synthesis and purification of a cross-linked oligonucleotide using denaturing polyacrylamide gel electrophoresis.
- Characterization of the cross-linked product to determine the site and nature of the adduct.
- Reaction of nitrogen mustard with various oligonucleotides to identify preferred cross-linking sequences.
Main Results:
- An interstrand cross-link was successfully created in a 5'-GAC-3'/3'-CTG-5' sequence using nitrogen mustard.
- The cross-link involved the N(7)-position of guanines on opposing strands, with stabilized imidazole ring-opened guanine adducts.
- Nitrogen mustard preferentially targets 5'-GXC-3' sequences for interstrand cross-linking, where X can be any base.
Conclusions:
- The study provides a method for creating site-specific DNA interstrand cross-links for further study.
- Nitrogen mustards exhibit sequence specificity in forming interstrand cross-links, favoring 5'-GXC-3' motifs.
- Understanding these preferences can inform the design of more targeted and effective DNA-damaging chemotherapeutic agents.