AbstractThe influence of 5‐bromo and 2′‐O‐methyl substitution of pyrimidine bases on nucleic acid triplex formation was examined by thermal denaturation and circular dichroism spectra with the following oligomers: 5′‐(Um) (I, Um= 2′‐O‐methyluridine), 5′‐(BUm)15T (II,BUm= 5‐bromo2′‐O‐metnyluridine), 5′‐(CmUm)6Cm‐T (III, Cm= 2′‐O‐methylcytidine), and 5′‐(CmUm)6CmT (IV). Both I and II form triplexes with dA16(A) or 5′‐d(C2A16C2)‐5′‐d(G2T16G2) (D), but III and IV form triplexes with 5′‐d(AG)8(G). Triplex formation involving two unmodified oligomers, dT16(T) and 5′‐d(CT)7(C), was used for comparison. The orders of melting temperatures (Tm) of triplexes were II A II (Tm= 48 °C)>I A I (34 °C)>T A T (22 °C); II D (Tm= 30 °C)>I D (22 °)>T D (12 °C); and IV G IV (Tm= 82 °C)>III G III (72 °C)>C G C (55 °C). The pKa of IVG IV, III G III, and C G C were determined by CD to be 6.5,6.3, and 6.2, respectively. Triplexes formed by oligonucleotides containing both 2′‐O‐methyl and 5‐bromo substitutions are more stable and have a greater pKa than those formed by oligonucleotides with 2′‐O‐methyl substitution only; which in turn a