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

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

Triplex-forming oligonucleotides trigger conformation changes of a target hairpin sequence.

We used a DNA duplex formed between the 5' end of a 69mer (69T) and an 11mer (OL7) as a substrate for BamHI. The former oligonucleotide folds into a hairpin structure, the stem of which contains a stretch of pyrimidines in one strand and consequently a stretch of purines in the other strand. The oligomer 69T was used as a target for complementary oligodeoxypyrimidines made of 10 nt (OL1), 16 nt (OL5) or 26 nt (OL2) which can engage the same 10 pyrimidine-purine-pyrimidine triplets with the 69T hairpin stem. Although the binding site of OL7 did not overlap that of OL1, OL2 or OL5, the BamHI activity on 69T-OL7 complexes was drastically modified in the presence of these triplex-forming oligomers: OL1 abolished the cleavage by BamHI whereas OL5 and OL2 strongly increased it. Using footprinting assays and point-mutated oligonucleotides we demonstrated that these variations were due to different conformations of the 69T-OL7 complex induced by the binding of oligomers OL1, OL2 or OL5. Therefore, oligonucleotides can act as structural switchers, offering one additional mode for modulating gene expression