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We have investigated the binding of a 26-mer antisense oligodeoxynucleotide to a 69-mer DNA hairpin with a 13 base pair stem, bearing an Rsa1 restriction site. The 5' part of the 26-mer annealed to a stretch of six purines at the bottom of the hairpin. The 3' part was designed to fold back to form a triplex with both the stem of the hairpin and with the sequence paired to its own 5' region. Using non-denaturing polyacrylamide gel electrophoresis, melting curves (Tm) and chemical footprinting, we were able to show the formation of a 'double-hairpin' complex between the 69-mer and the 26-mer antisense oligopyrimidines. The association was both sequence and pH-dependent. The formation of a double hairpin complex was shown to prevent the alkylation of the 69-mer DNA target by an oligonucleotide-nitrogen mustard conjugate and to selectively inhibit the action of Rsa1.

The binding of an antisense oligonucleotide to a hairpin structure via triplex formation inhibits chemical and biological reactions