Double hairpin complexes allow accommodation of all four base pairs in triple helices containing both DNA and RNA strands.

We investigated the binding of an antisense oligodeoxynucleotide to a stem-loop structure corresponding to the mini-exon sequence of the protozoan parasite Leishmania amazonensis. This oligomer was designed to anneal to the single-stranded region adjacent to the bottom of the hairpin and to fold back on itself, giving rise to a "double-hairpin" complex that involved a local triplex. This imposed the recognition, by the third strand, of a "purine" strand containing 6 interspersed pyrimidines out of 15 nucleic acid bases. The sequence of the complementary oligonucleotide was derived from the so-called pyrimidine motif; the third strand of the anti-mini-exon oligomer was parallel to the purine strand of the target. Electrophoretic mobility shift assays and footprinting studies demonstrated that such an antisense oligomer was able to bind to both the DNA and RNA versions of the Leishmania hairpin. These double hairpin complexes allowed the formation at pH 6.0 of a triple-stranded structure, despite the presence of 4 A:TG and 2 G:CT triplets out of 15.