Design of a nonnatural deoxyribonucleoside for recognition of GC base pairs by oligonucleotide-directed triple helix formation

The deoxyribonucleoside 1-(2-deoxy-beta-D-ribofuranosyl)-3-methyl-5-amino-1H-pyrazolo4,3-d]py rimidin-7-one (P1) was designed such that two specific hydrogen bonds would form with guanine (G) of a Watson-Crick guanine-cytosine (GC) base pair in the major groove of double-helical DNA. One edge of the P1 heterocycle mimics N3-protonated cytosine, which would circumvent the pH dependence observed for the formation of triple helices containing C + GC base triplets. P1 was synthesized in five steps and incorporated by automated methods in pyrimidine oligodeoxyribonucleotides. From affinity cleaving analyses, the stabilities of base triplets decrease in the order P1.GC >> P1.CG >> P1.AT approximately P1.TA (pH 7.4, 35-degrees-C). P1 binds GC base pairs within a pyrimidinte triple-helix motif as selectively and strongly as C but over an extended pH range. Oligodeoxyribonucleotides containing P1 residues were shown to bind within plasmid DNA a single 15 base pair site containing five GC base pairs at pH 7.8 and a single 16 base pair site containing six contiguous GC base pairs at pH 7.4.