title: Minimum number of 2'-O-(2-aminoethyl) residues required for gene knockout activity by triple helix forming oligonucleotides.
abstract: Triple helix forming oligonucleotides (TFOs) that bind chromosomal targets in living cells may become tools for genome manipulation, including gene knockout, conversion, or recombination. However, triplex formation by DNA third strands, particularly those in the pyrimidine motif, requires nonphysiological pH and Mg(2+) concentration, and this limits their development as gene-targeting reagents. Recent advances in oligonucleotide chemistry promise to solve these problems. For this study TFOs containing 2'-O-methoxy (2'-OMe) and 2'-O-(2-aminoethyl) (2'-AE) ribose substitutions in varying proportion have been constructed. The TFOs were linked to psoralen and designed to target and mutagenize a site in the hamster HPRT gene. T(m) analyses showed that triplexes formed by these TFOs were more stable than the underlying duplex, regardless of 2'-OMe/2'-AE ratio. However, TFOs with 2'-AE residues were more stable in physiological pH than those with only 2'-OMe sugars, as a simple function of 2'-AE content. In contrast, gene knockout assays revealed a threshold requirement--TFOs with three or four 2'-AE residues were at least 10-fold more active than the TFO with two 2'-AE residues. The HPRT knockout frequencies with the most active TFOs were 300-400-fold above the background, whereas there was no activity against the APRT gene, a monitor of nonspecific mutagenesis.
authors.affiliation: Laboratory of Molecular Gerontology, National Institutes on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
authors.forename: Nitin
authors.initials: N
authors.lastname: Puri
authors.forename: Alokes
authors.initials: A
authors.lastname: Majumdar
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: Andre
authors.initials: A
authors.lastname: Boyd
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; D006224:Cricetinae; D003412:Cricetulus; D003596:Cytosine / Q000031:analogs & derivatives / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D018274:Electroporation; D018390:Gene Targeting / Q000379:methods*; D006358:Hot Temperature; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D012266:Ribose / Q000031:analogs & derivatives / Q000737:chemistry; D013936:Thymidine / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D009841:Oligonucleotides; C051802:triplex DNA; D012266:Ribose; D003596:Cytosine; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D013936:Thymidine