Acid-Base and Metal-Ion Binding Properties of the RNA Dinucleotide Uridylyl-(5′→3′)-[5′]uridylate (pUpU3−)

It is well known that Mg2+ and other divalent metal ions bind to the phosphate groups of nucleic acids. Subtle differences in the coordination properties of these metal ions to RNA, especially to ribozymes, determine whether they either promote or inhibit catalytic activity. The ability of metal ions to coordinate simultaneously with two neighboring phosphate groups is important for ribozyme structure and activity. However, such an interaction has not yet been quantified. Here, we have performed potentiometric pH titrations to determine the acidity constants of the protonated dinucleotide H2(pUpU)−, as well as the binding properties of pUpU3− towards Mg2+, Mn2+, Cd2+, Zn2+, and Pb2+. Whereas Mg2+, Mn2+, and Cd2+ only bind to the more basic 5′‐terminal phosphate group, Pb2+, and to a certain extent also Zn2+, show a remarkably enhanced stability of the [M(pUpU)]− complex. This can be attributed to the formation of a macrochelate by bridging the two phosphate groups within this dinucleotide by these metal ions. Such a macrochelate is also possible in an oligonucleotide, because the basic structural units are the same, despite the difference in charge. The formation degrees of the macrochelated species of [Zn(pUpU)]− and [Pb(pUpU)]− amount to around 25 and 90 %, respectively. These findings are important in the context of ribozyme and DNAzyme catalysis, and explain, for example, why the leadzyme could be selected in the first place, and why this artificial ribozyme is inhibited by other divalent metal ions, such as Mg2+. Metal ions are key to ribozyme activity: Simultaneous coordination of a metal ion to neighboring phosphate groups (see figure) is a key feature of ribozyme structure and function. The ability of different metal ions to form such a macrochelate was quantified by using the dinucleotide pUpU3−. The results show that, for example, Mg2+ binds mainly in a monodentate and Pb2+ in a bidentate fashion.