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

Abstract

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 anti-sense 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:T*G and 2 G:C*T triplets out of 15.