title: Cell cycle modulation of gene targeting by a triple helix-forming oligonucleotide.
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 approximately 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.
authors.affiliation: Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
authors.forename: Alokes
authors.initials: A
authors.lastname: Majumdar
authors.forename: Nitin
authors.initials: N
authors.lastname: Puri
authors.forename: Bernard
authors.initials: B
authors.lastname: Cuenoud
authors.forename: Francois
authors.initials: F
authors.lastname: Natt
authors.forename: Pierre
authors.initials: P
authors.lastname: Martin
authors.forename: Alexander
authors.initials: A
authors.lastname: Khorlin
authors.forename: Natalia
authors.initials: N
authors.lastname: Dyatkina
authors.forename: Albert J
authors.initials: AJ
authors.lastname: George
authors.forename: Paul S
authors.initials: PS
authors.lastname: Miller
authors.forename: Michael M
authors.initials: MM
authors.lastname: Seidman
mesh_terms: D000818:Animals; D016466:CHO Cells; D002453:Cell Cycle; D006224:Cricetinae; D004247:DNA / Q000502:physiology*; D018390:Gene Targeting; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics
chemical_list: C051802:triplex DNA; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase