Targeted generation of DNA strand breaks using pyrene-conjugated triplex-forming oligonucleotides.
Gene targeting by triplex-forming oligonucleotides (TFOs) has proven useful for gene modulation in vivo. Photoreactive molecules have been conjugated to TFOs to direct sequence-specific damage in double-stranded DNA. However, the photoproducts are often repaired efficiently in cells. This limitation has led to the search for sequence-specific photoreactive reagents that can produce more genotoxic lesions. Here we demonstrate that photoactivated pyrene-conjugated TFOs (pyr-TFOs) induce DNA strand breaks near the pyrene moiety with remarkably high efficiency and also produce covalent pyrene-DNA adducts. Free radical scavenging experiments demonstrated a role for singlet oxygen activated by the singlet excited state of pyrene in the mechanism of pyr-TFO-induced DNA damage. In cultured mammalian cells, the effect of photoactivated pyr-TFO-directed DNA damage was to induce mutations, in the form of deletions, approximately 7-fold over background levels, at the targeted site. Thus, pyr-TFOs represent a potentially powerful new tool for directing DNA strand breaks to specific chromosomal locations for biotechnological and potential clinical applications.
Benfield
Aaron P
AP
Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957, USA.
Macleod
Michael C
MC
Liu
Yaobin
Y
Wu
Qi
Q
Wensel
Theodore G
TG
Vasquez
Karen M
KM
D004249:DNA Damage / Q000187:drug effects*; D016166:Free Radical Scavengers; D007700:Kinetics; D016296:Mutagenesis; D009154:Mutation; D009841:Oligonucleotides / Q000494:pharmacology*; D010777:Photochemistry; D011721:Pyrenes / Q000494:pharmacology*; D017384:Sequence Deletion / Q000187:drug effects
D016166:Free Radical Scavengers; D009841:Oligonucleotides; D011721:Pyrenes