Recognition of all four base pairs of double-helical DNA by triple-helix formation: design of nonnatural deoxyribonucleosides for pyrimidine•purine base pair binding

The sequence-specific recognition of double-helical DNA by oligonucleotide-directed triple-helix formation is limited mostly to purine tracts. Design leads that could expand the recognition code to all four Watson-Crick base pairs would provide one step toward a general solution targeting single sites in megabase size DNA. The nonnatural deoxyribonucleoside 1-(2-deoxy-beta-D-ribofuranosyl)-4-(3-benzamidophenyl)imidazole (D3) was synthesized in four steps and incorporated by automated methods into pyrimidine oligodeoxyribonucleotides. Within a pyrimidine oligonucleotide, D3 binds pyrimidine.purine base pairs with higher affinity than it binds purine.pyrimidine base pairs. From affinity-cleaving analysis, the stabilities of base triplets decrease in the order D3.TA is similar to D3.CG > D3.AT > D3.GC. Such specificity allows binding by triple-helix formation at an 18 base pair site in SV40 DNA containing all four base pairs at physiologically relevant pH and temperature. The stabilities of these novel triplets may be an example of shape-selective recognition of CG and TA Watson-Crick base pairs in the major groove