A nonnatural nucleoside, 1-(2-deoxy-beta-D-ribofuranosyl)-3-methyl-5-amino-1H-pyrazolo [4,3-d]pyrimidin-7-one (P), mimics protonated cytosine and specifically binds GC base pairs within a pyrimidine . purine . pyrimidine triple helix. Quantitative footprint titration experiments at neutral pH (22 degrees C, 100 mM NaCl, 10 mM bis-tris, 250 mu M spermine) reveal dramatic sequence composition effects on the energetics of triple helix formation by oligonucleotides containing P or 5-methylcytosine (C-m). Purine tracts of sequence composition 5’-d(AAAAA-GAGAGAGAGA)-3’ are bound by oligonucleotide 5’-d((TTTTTCTCTCTCTCT)-C-m-C-m-C-m-C-m-C-m)-3’4 orders of magnitude more strongly than by 5’-d(TTTTTPTPTPTPTPT)-3’ (K-T approximate to 3 X 10(9) M(-1) and K-T = 1 X 10(5) M(-1), respectively). Conversely, purine tracts of sequence composition 5’-d(AAAAGAAAAGGGGGGA)-3’ are bound by oligonucleotide 5’-d((TTTTCTTTTCCCCCCT)-C-m-C-m-C-m-C-m-C-m-C-m-C-m)-3’5 orders of magnitude less strongly than by 5’-d((TTTTCTTTTPPPPPPT)-C-m)-3’ (K-T < 5 x 10(4) M(-1) and K-T approximate to 4 X 10(9) M(-1), respectively). The complementary nature of P and C-m expands the repertoire of G-rich sequences which may be targeted by triple helix formation.