We are developing a gene targeting program based on the capacity of oligonucleotides to form stable triple helix complexes with specific sequences 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 structure of mammalian chromatin 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. 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. We also find that the targeting oligonucleotides can be used to direct homologous recombination. Cells treated with the TFO and a donor DNA with homology to the region around the target sequence show knock in of the donor DNA at frequencies 3-4 orders of magnitude above that seen with cells treated with donor alone.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Intramural Research (Z01)
Project #
1Z01AG000738-05
Application #
6815326
Study Section
(LMG)
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2003
Total Cost
Indirect Cost
Name
Aging
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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
Cheng, Wen-Hsing; von Kobbe, Cayetano; Opresko, Patricia L et al. (2004) Linkage between Werner syndrome protein and the Mre11 complex via Nbs1. J Biol Chem 279:21169-76
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

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