Cell cycle modulation of gene targeting by a triple helix-forming oligonucleotide

Alokes Majumdar, Nitin Puri, Bernard Cuenoud, Francois Natt, Pierre Martin, Alexander Khorlin, Natalia Dyatkina, Albert J. George, Paul S. Miller, Michael M. Seidman

Research output: Contribution to journalArticle

Abstract

Successful gene-targeting reagents must be functional under physiological conditions and must bind chromosomal target sequences embedded in chromatin. Triple helix-forming oligonucleotides (TFOs) recognize and bind specific sequences via the major groove of duplex DNA and may have potential for gene targeting in vivo. We have constructed chemically modified, psoralen-linked TFOs that mediate site-specific mutagenesis of a chromosomal gene in living cells. Here we show that targeting efficiency is sensitive to the biology of the cell, specifically, cell cycle status. Targeted mutagenesis was variable across the cycle with the greatest activity in S phase. This was the result of differential TFO binding as measured by cross-link formation. Targeted cross-linking was low in quiescent cells but substantially enhanced in S phase cells with adducts in ∼20-30% of target sequences. 75-80% of adducts were repaired faithfully, whereas the remaining adducts were converted into mutations (>5% mutation frequency). Clones with mutations could be recovered by direct screening of colonies chosen at random. These results demonstrate high frequency target binding and target mutagenesis by TFOs in living cells. Successful protocols for TFO-mediated manipulation of chromosomal sequences are likely to reflect a combination of appropriate oligonucleotide chemistry and manipulation of the cell biology.

Original languageEnglish (US)
Pages (from-to)11072-11077
Number of pages6
JournalJournal of Biological Chemistry
Volume278
Issue number13
DOIs
Publication statusPublished - Mar 28 2003

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Majumdar, A., Puri, N., Cuenoud, B., Natt, F., Martin, P., Khorlin, A., ... Seidman, M. M. (2003). Cell cycle modulation of gene targeting by a triple helix-forming oligonucleotide. Journal of Biological Chemistry, 278(13), 11072-11077. https://doi.org/10.1074/jbc.M211837200