High-affinity triple helix formation by synthetic oligonucleotides at a site within a selectable mammalian gene.
Specific recognition of duplex DNA by a single-stranded oligonucleotide via the formation of triplex DNA is a rational approach for targeting specific regions of a genome. By screening a number of potential target sites for triple helix formation within mammalian genes that allow genetic selection in cell culture, we have identified a site within intron 1 of the hamster adenine phosphoribosyltransferase (APRT) gene that specifically binds a triplex-forming oligodeoxyribonucleotide (TFO) with high affinity. Under optimal conditions for triplex formation, the equilibrium dissociation constant is in the nanomolar range (Kd = 7 x 10(-10) M). This high-affinity binding is very specific, as a 10(5)-fold excess of genomic DNA reduced triplex formation less than 10-fold, and within a 6928-bp plasmid bearing the APRT gene, only restriction fragments containing the intron 1 site were found to bind the TFO. Results of DNase I protection assays were consistent with the TFO binding in an antiparallel orientation via reverse Hoogsteen hydrogen bonds in the major groove of the duplex. We have examined the kinetics of triplex formation as well as the effects of ionic composition and chemical modifications of the TFO on triplex formation. While divalent cations were not required for triplex formation, Mg2+ stabilized the triplex apparently through inhibition of TFO dissociation, with a mean bound lifetime of > 17 h for the triplex at Mg2+ concentrations above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
Vasquez
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Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
Wensel
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Hogan
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Wilson
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D000228:Adenine Phosphoribosyltransferase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D006224:Cricetinae; D004247:DNA / Q000138:chemical synthesis* / Q000378:metabolism; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; C051802:triplex DNA; D004247:DNA; D000228:Adenine Phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase