Triple helix formation of oligodeoxynucleotides (ODNs) with a 15 base pair poly-purine DNA target in the HER2 promoter was examined by footprinting analysis. 7-deaza-2'-deoxyxanthosine (dzaX) was identified as a purine analogue of thymidine (T) which forms dzaX:A-T triplets. ODNs containing 2'-deoxyguanosine (G) and dzaX were found to form triple helices in an anti-parallel orientation, with respect to the poly-purine strand of the target DNA. In comparative studies under physiological K+ and Mg++ concentrations and at pH 7.2, the ODNs containing G and dzaX showed high affinity to the target sequence while the ODNs containing G and T were not able to bind. In the absence of added monovalent salts both ODNs showed high affinity to the target sequence. The substitution of 7-deaza-2'-deoxyguanosine for G substantially decreased the capacity of the ODNs to form triple helices under physiological conditions, indicating that dzaX may be unique in its ability to enhance triple helix formation in the anti-parallel motif.