We are developing a gene targeting program based on the capacity of oligonucleotides to form stable triple helix complexes with specific sequence in duplex DNA. This approach has the promise to become a simple and efficient technology for delivering DNA reactive compounds to specific sites in chromosomal DNA in living cells. Applications include gene knockout, directed gene conversion and recombination, and, perhaps, gene therapy. Triple helices have been known for over 40 years and have been the subject of many studies in vitro. However there is direct evidence that the protein:nucleic acid structure of mammalian chromosomes would preclude access to triplex forming oligos (TFO). We prepared a TFO linked to a photoactivatable DNA mutagen directed against a sequence in a gene (HPRT) frequently used as a mutation reporter. We introduced this into mammalian cells and, after photoactivation of the mutagen, isolated colonies of cells with mutations in the target gene. Sequence analysis showed that the mutations were located at the target sequence within the gene. We have prepared TFOs with novel sugar modifications that show enhanced targeting activity. Treatment of S phase cells with these TFOs results in 30% of targeted crosslinking and 5-10% mutation frequencies, while both crosslinking and mutagenesis are much lower in quiescent cells. These results indicate that the accessibility of chromosomal target sites in mammalian cells is modulated by the biology of the cell. Furthermore the frequency of mutagenesis is sufficiently high to allow identification of colonies with sequence changes in simple screens of a few clones.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Intramural Research (Z01)
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Peng, Xiaohua; Li, Hong; Seidman, Michael (2010) A Template-Mediated Click-Click Reaction: PNA-DNA, PNA-PNA (or Peptide) Ligation, and Single Nucleotide Discrimination. European J Org Chem 2010:4194-4197
Alam, Md Rowshon; Majumdar, Alokes; Thazhathveetil, Arun Kalliat et al. (2007) Extensive sugar modification improves triple helix forming oligonucleotide activity in vitro but reduces activity in vivo. Biochemistry 46:10222-33
Shirley, Thomas L; Lewers, J Chris; Egami, Kiyoshi et al. (2007) A human neuronal tissue culture model for Lesch-Nyhan disease. J Neurochem 101:841-53
Shahid, Kazi Abdus; Majumdar, Alokes; Alam, Rowshon et al. (2006) Targeted cross-linking of the human beta-globin gene in living cells mediated by a triple helix forming oligonucleotide. Biochemistry 45:1970-8
Cheng, Wen-Hsing; Kusumoto, Rika; Opresko, Patricia L et al. (2006) Collaboration of Werner syndrome protein and BRCA1 in cellular responses to DNA interstrand cross-links. Nucleic Acids Res 34:2751-60
Richards, Sally; Liu, Su-Ting; Majumdar, Alokes et al. (2005) Triplex targeted genomic crosslinks enter separable deletion and base substitution pathways. Nucleic Acids Res 33:5382-93
Kalish, Jennifer M; Seidman, Michael M; Weeks, Daniel L et al. (2005) Triplex-induced recombination and repair in the pyrimidine motif. Nucleic Acids Res 33:3492-502
Seidman, Michael M; Puri, Nitin; Majumdar, Alokes et al. (2005) The development of bioactive triple helix-forming oligonucleotides. Ann N Y Acad Sci 1058:119-27
Opresko, Patricia L; Otterlei, Marit; Graakjaer, Jesper et al. (2004) The Werner syndrome helicase and exonuclease cooperate to resolve telomeric D loops in a manner regulated by TRF1 and TRF2. Mol Cell 14:763-74
Seidman, Michael M (2004) Oligonucleotide mediated gene targeting in mammalian cells. Curr Pharm Biotechnol 5:421-30

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