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Sequence-specific DNA double-strand breaks induced by triplex forming 125I labeled oligonucleotides
1Department of Nuclear Medicine, Clinical Center, National Institutes of Health, Bethesda, MD 20892.
Nucleic Acids Research
|November 25, 1994
Summary
Radioactive decay of Iodine-125 within a triplex-forming oligonucleotide (TFO) specifically induces double-strand breaks in the Human Immunodeficiency Virus (HIV) nef gene. This targeted DNA damage occurs only when the TFO forms a triplex structure.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- The Human Immunodeficiency Virus (HIV) relies on genes like nef for its replication and pathogenesis.
- Targeting viral DNA with sequence-specific agents offers a potential therapeutic strategy.
- Oligonucleotide-based approaches, such as triplex-forming oligonucleotides (TFOs), can bind to specific DNA sequences.
Purpose of the Study:
- To investigate the potential of using a radiolabeled TFO to induce sequence-specific DNA damage within the HIV nef gene.
- To determine if the radioactive decay of an incorporated isotope can cause double-strand breaks (DSBs) in a targeted manner.
Main Methods:
- A TFO complementary to a polypurine-polypyrimidine region of the HIV nef gene was synthesized.
- The TFO was labeled with Iodine-125 (125I) at a specific deoxycytosine residue.
- The 125I-labeled TFO was incubated with a plasmid containing the HIV nef gene fragment under conditions favoring triplex formation.
- Control experiments included incubation under conditions preventing triplex formation and using a TFO labeled with Phosphorus-32 (32P).
- Double-strand break sites were mapped with single base resolution.
Main Results:
- The decay of 125I within the TFO induced sequence-specific DSBs in the nef gene fragment when triplex formation occurred.
- No DSBs were observed when triplex formation was inhibited by altering ionic conditions.
- No DSBs were detected when the TFO was labeled with 32P, indicating the effect is specific to 125I decay.
- DSB sites were localized within 10 base pairs of the 125I-labeled deoxycytosine residue in the TFO.
- Approximately one DSB was produced per 125I decay event.
Conclusions:
- Radiolabeled TFOs, specifically those incorporating 125I, can function as agents for targeted induction of DNA double-strand breaks in specific gene sequences.
- This mechanism demonstrates a sequence-specific DNA damaging capability mediated by radioactive decay within a TFO-DNA triplex.
- The findings suggest a potential novel strategy for targeting viral genomes, such as that of HIV, using radiolabeled oligonucleotides.