The thermal stability of the parallel motif triple helix DNA containing CG.CH + base triplets is improved by silver ions. The N3 protons of cytosines on the third strand are replaced by the ions to form CG.CAg + , which are stable regardless of the pH values. The activity of the DNAzyme that binds its substrate DNA through triple helix formation was examined in the presence of silver ions. The DNAzyme activity was retained even at neutral pH with the assistance of silver ions.Ribozymes and DNAzymes are a class of functional nucleic acids obtained by molecular evolution. The parallel motif structure of a DNA triplex containing CG.CH + base triplets is stable only under weak acidic conditions, because the N3 position of the cytosines (pK a μ 4.5) on the third strand needs to be protonated for complementarity with the Hoogsteen side of guanines.11 Previously, we reported that silver ions stabilized the parallel motif structure of a DNA triplex even under weak basic conditions.12 The N3 protons of CG.CH + base triplets were quantitatively substituted by silver ions to form silver ion-mediated stable base triplets, CG.CAg + ( Figure 1a). Judging from the experimental conditions, the binding affinity of the silver ion was rather high; the apparent dissociation constant of the silver ion from the binding pocket in CG.C seemed to be less than 10 ¹6 M ¹1. Several applications have been made for some analytical and engineering purposes. The molecular beacon 13 and DNA array 14 that targets the specific sequences in duplexes were proposed based on the stable triplex formation in the presence of silver ions. Luminous Ag clusters were successfully prepared using the DNA triplex as a template, on which silver ions were allocated at defined position as CG.CAg + . 15Recently, specific interactions with certain metal ions have been used for regulating the functions of biomolecules. Here, we intended to regulate the activity of a DNA-cleaving DNAzyme by silver ions. The DNAzyme used in this study was Cu 2+ -dependent and first reported by Breaker et al. 17 The DNAzyme consists of a hairpin duplex and a short singlestranded regions for capturing single-stranded DNA substrates. Thus, the DNAzyme binds the substrate DNA through the cooperative formation of a triple helix and a short duplex. Silver ions were found to significantly stabilize the triple helix of the parallel motif.12a Therefore, the pH profile of the catalytic activity of the DNAzyme would be altered by the addition of silver ions through specific interactions with CG.C base triplets. The sequences of the DNAs used in this study are shown in Figure 1b. Two DNAzymes Dz1 and Dz2 carrying different hairpin-arm sequences and the substrate for Dz1 (S) were prepared to examine the substrate binding specificity through triplex formation.18 Figure 2 shows the results of UV melting experiments of the complex, DNAzyme-substrate (Dz1-S), in the absence (a) and presence (b) of silver ions. The experiments were carried out in buffer solutions at three different pH values (50-mM MES...