Oligonucleotide-directed triple helix formation is one of the most powerful methods for the sequence-specific recognition of double-helical DNA. 1·* 12 34Pyrimidine oligonucleotidesbind purine tracts in the major groove of DNA parallelto the purine Watson-Crick strand through the formationof specific Hoogsteen-type hydrogen bonds. 1-4 Specificity is derived from thymine (T) recognition of adenine· thy mine (·) base pairs (·· triplet) and N3-protonated cytosine (C+) recognition of guanine-cytosine (GC) base pairs (C+ GC triplets). 1-4 The sequence-specific recognition of double-helical DNA by a third strand to form a triple helix is limited to mostly purine tracts. Although G in the third strand has been found to specifically bind to·, 5 67the lower stability of the GTA triplet and its dependence on the sequence of the neighboring triplets reveals that this will have limitations. 5-8 In an attempt to extend the recognition code to all four Watson-Crick base pairs, the nonnatural deoxyribonucleoside 1-(2-deoxy-0-D-ribofuranosyl)-4-(3-benzamido) phenylimidazole [D3] 9 was synthesized and incorporated into pyrimidine DNA oligonucleotides (Figure la). It was found that D3 selectively recognizes both· and CG Watson-Crick base pairs within the pyrimidine-purine-pyrimidine triple-helix motif. 9 This was also found to have a nearest neighbor dependence. 5b In orderto determine the binding mode of the nonnatural deoxyribonucleoside D3 with the Watson-Crick base pair·,