The use of oligodeoxynucleotides (ODNs) to control gene expression at the DNA level is hindered by sequence recognition strategies. Triplex formation by Hoogsteen hydrogen bonding of the ODN to purines in a homopurine run1 is most often used, but this critical target sequence requirement limits the applicability of this approach. Ideally, one would like to be able to target any desired site in DNA. Although RecA protein can catalyze binding of ODNs to complementary sequences in DNA, 2 this method faces challenges for implementation in cells. The versatility of Watson-Crick sequence targeting could be realized by displacement loop (D-loop) formation, which, in principle, places no limits on targeting sequence. It does, however, present significant thermodynamic and kinetic barriers. 3 These might be overcome if (a) the rate of D-loop formation could be accelerated and (b) the D-loop could be stabilized.

DNA strand invasion has been observed with homopyrimidine peptide nucleic acids (PNA) at DNA homopurine runs, 4 where tightly bound PNA2-DNA complexes are formed. Certain PNA oligomers can directly invade DNA to give Watson-Crick duplexes. 5 ODNs targeted to a cruciform in supercoiled DNA form stable D-loops, 6 and D-loop formation can be promoted by a nuclease conjugated to the ODN. 7 Additionally, Kobets et al. have shown that certain areas of chromatin may be available to Watson-Crick recognition and that this modification is eliminated by pretreatment with S1 nuclease. 8 We sought to induce D-loop formation in double stranded DNA using phosphodiester-based oligonucleotides and report here a new method for doing so. This method uses ODNs containing a triplex-forming guide sequence