Targeting of single-stranded DNA and RNA containing adjacent pyrimidine and purine tracts by triple helix formation with circular and clamp oligonucleotides

The aim of this work was to construct an anti-messenger targeted to the pim -1 oncogene transcript, based on circular or clamp oligodeoxyribonucleotide s. The formation of bimolecular triplexes by clamp or circular oligonucleot ides was investigated using single-stranded targets of both DNA (5'-CCCTCCT TTGAAGAA-3') and RNA type (5'-CCCUCCUUUGAAGAA-3'). The third, 'Hoogsteen' s trand of the triplex was represented by G,T-rich sequences. The secondary s tructures of the complexes were determined by thermal denaturation, circula r dichroism and gel mobility shift experiments and shown to depend on the n ature of the target strand. With DNA as target, the sequence of a clamp (or circular) oligonucleotide that formed the triple helix was 3'-<(GGGAGGAAAC TTCTT)under bar>TT-TTGTTGTTT-TT-GGTGGG-5', where the first TT dinucleotide (in italics) is a linker and the second TT (bold) represents the bridge thr ough which the 'Hoogsteen' strand switches from one strand of the Watson-Cr ick duplex to the other, once the duplex is formed by the corresponding por tion of the anti-messenger (underlined). The portion of the 'Hoogsteen' seq uence of the triplex between the two TT dinucleotides binds to the 3' extre mity of the target strand and runs parallel to it. The portion situated at the 5' end of the oligonucleotide switches to the purine tract of the compl ementary strand of the duplex and is antiparallel to it. In contrast, with RNA as target, for a branched clamp oligonucleotide that formed a triple he lix over its entire length (5'-<(TTCTTCAAAGGAGGG)under bar>-30'(boolean AND )3'-GGGTGGTTT-T-GTTGTT-5') the portion of the 'Hoogsteen' sequence that bou nd to the 3' extremity of the target strand had to be antiparallel to it.