title: Enhancement and inhibition by 2'-O-hydroxyethyl residues of gene targeting mediated by triple helix forming oligonucleotides.
abstract: Reagents that recognize and bind specific genomic sequences in living mammalian cells would have great potential for genetic manipulation, including gene knockout, strain construction, and gene therapy. Triple helix forming oligonucleotides (TFOs) bind specific sequences via the major groove, but pyrimidine motif TFOs are limited by their poor activity under physiological conditions. Base and sugar analogues that overcome many of these limitations have been described. In particular, 2'-O-modifications influence sugar pucker and third strand conformation, and have been important to the development of bioactive TFOs. Here we have analyzed the impact of 2'-O-hydroxyethyl (2'-HE) substitutions, in combination with other 2' modifications. We prepared modified TFOs conjugated to psoralen and measured targeting activity in a gene knockout assay in cultured hamster cells. We find that 2'-HE residues enhance the bioactivity of TFOs containing 2'-O-methyl (2'-OMe) modifications, but reduce the bioactivity of TFOs containing, in addition, 2'-O-aminoethyl (2'-AE) residues.
authors: Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
authors: Mrinalkanti
authors: M
authors: Kundu
authors: Fumi
authors: F
authors: Nagatsugi
authors: Alokes
authors: A
authors: Majumdar
authors: Paul S
authors: PS
authors: Miller
authors: Michael M
authors: MM
authors: Seidman
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004247:DNA / Q000737:chemistry* / Q000494:pharmacology*; D018390:Gene Targeting / Q000379:methods*; D006900:Hydroxylation; D015394:Molecular Structure; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000737:chemistry* / Q000494:pharmacology*; D013866:Thioguanine / Q000494:pharmacology
chemical_list: D009841:Oligonucleotides; C051802:triplex DNA; D004247:DNA; D013866:Thioguanine