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DNA Cleavage by ¹¹¹In-Labeled Oligodeoxyribonucleotides

Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland
Epoch Pharmaceuticals, Inc., Bothell, Washington
Novosibirsk Institute of Bioorganic Chemistry, Novosibirsk, Russia

Correspondence: For correspondence or reprints contact: Valeri N. Karamychev, MD, Visiting Fellow, NMD, CC, National Institutes of Health, 10 Center Dr., MSC 1180, Bethesda, MO 20892-1180.

ABSTRACT

We studied the fine structure of DNA damage produced by the decay of ¹¹¹In incorporated into duplex and triplex DNA strands to evaluate the usefulness of this radionuclide for sequence-specific DNA cleavage. Methods: Oligodeoxyribonucleotides (ODNs) were prepared with ¹¹¹In attached by diethylenetriamine-pentaacetic acid (DTPA) at the 5' end or 3' end through a long chemical linker or to an internal nucleotide position through a short linker. Subsequent formation of DNA duplexes and triplexes was confirmed by gel electrophoresis. The ¹¹¹In-induced breaks were assayed in denaturing polyacrylamide gel electrophoresis with a single-nucleotide resolution. Results: ¹¹¹In-labeled oligonucleotides of high specific activity (740-1554 TBq/mmol) were synthesized. The presence of the bulky ¹¹¹In-DTPA group did not impede duplex or triplex formation. Localized DNA breaks were observed in all duplexes and triplexes formed. The majority of DNA breaks in duplex formations were located within ±10 nucleotides from the site of attachment of the ¹¹¹In-bearing linker. The yield of DNA breaks per decay was 0.38 in a duplex with internally modified ODNs. This is nearly 2 times less than the yield of DNA breaks in the same duplex with ¹²⁵I attached through the same linker. The yield of DNA breaks in the pyrimidine and purine strands of DNA triplexes with ¹¹¹In attached to the triplex-forming ODNs through the linkers of different length varied from 0.05 to 0.10. The distribution of DNA breaks was wider in comparison with the duplex experiment. The lower yields of breaks per ¹¹¹In decay compared with ¹²⁵I may be not only the result of lower deposited energy but also of the ionic repulsion of the negatively charged ¹¹¹In-DTPA group from the DNA strands. Conclusion: We have shown that decay of ¹¹¹In produces highly localized DNA breaks. ¹¹¹In introduced into triplex- and duplex-forming ODNs through hydrocarbon linkers produces sequence-specific DNA strand breaks with an efficiency nearly comparable with that of ¹²⁵I. These findings are supportive of our proposed use of ¹¹¹In-ODNs for gene-specific radiotherapy.

Key Words: Auger electrons • ¹¹¹In • DNA double-strand breaks

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