Recently, studies of sequence-specific triplex formation of short synthetic oligonucleotides (or their analogues) have been of prime interest. 1" 6 In this report, a synthesis of a new oligonucleotide analogue containing2-amino-5-(2-0-methyl-/3-D-ribo-furanosyl)-4 (l//)-pyrimidinone (2'-0-methylpseudoisocytidine or 1) in substitution for 2'-deoxycytidine and the triplex formation of this oligonucleotide analogue with a target duplex at neutral pH are reported. A triplex can be formed as a triad consisting of a homo-pyrimidine strand, a homopurine strand, and a homopyrimidine strand. 7" 11 The third strand (ie, the second pyrimidine strand) is located in the major groove of a duplex with Watson-Crick base pairing. 12" 16 Thymines or cytosines in the third strand form Hoogsteen type hydrogen bondings with adenines or guanines in the homopurine strand, respectively. Since protonation of cytosine bases in acidic conditions is essential in order to provide the second hydrogen bonding between the protonated cytosine and guanine in the Hoogsteen pair of the triad (Figure 1, upper-left panel), this CG-C+ triad is unstable in physiological conditions. 17" 19 This requirement limits the formation of triplex in living cells since the cellular pH is usually above pH 7.0. To overcome this limitation, we designed and synthesized an