Four base recognition by triplex-forming oligonucleotides at physiological pH

We have achieved recognition of all 4 bp by triple helix formation at physiological pH, using triplex-forming oligonucleotides that contain four different synthetic nucleotides. BAU [2'-aminoethoxy-5-(3-aminoprop-1ynyl) uridine] recognizes AT base pairs with high affinity, P-Me (3-methyl-2 aminopyridine) binds to GC at higher pHs than cytosine, while (PP)-P-A (6-(3- aminopropyl)7- methyl-3H-pyrrolo[ 2,3-d] pyrimidin-2(7H)-one) and S [N-(4-(3- acetamidophenyl) thiazol-2-yl-acetamide)] bind to CG and TA base pairs, respectively. Fluorescence melting and DNase I footprinting demonstrate successful triplex formation at a 19mer oligopurine sequence that contains two CG and two TA interruptions. The complexes are pH dependent, but are still stable at pH 7.0. BAU, P-Me and (PP)-P-A retain considerable selectivity, and single base pair changes opposite these residues cause a large reduction in affinity. In contrast, S is less selective and tolerates CG pairs as well as TA.