Inhibition of replication initiation by triple helix-forming oligonucleotides

Oligonucleotide-directed triple helix formation constitutes a new approach to block gene expression via transcription inhibition. In addition triple helices might inhibit replication. We have examined the capacity of triple helix-forming oligonucleotides to inhibit the initiation of replication on a single-stranded DNA template using T7 DNA polymerase (Sequenase). We show that triple helix formation at the primer initiation site efficiently inhibits DNA polymerization, by preventing binding of the polymerase. The effect is dependent on the distance between the 3'-end of the primer and the triple helix boundary. Inhibition becomes ineffective when this distance is greater than 3 nucleotides. The presence of three base-pairs outside the triple-helical region on the 3'-side of the primer is therefore sufficient to allow for initiation of DNA replication.