Differential binding of Mg²⁺, Zn²⁺, and Cd ²⁺ at two sites in a hammerhead ribozyme motif, determined by ¹⁵N NMR

A decamer duplex model of Domain II of the hammerhead ribozyme was synthesized with [8-¹³C-1,7,NH₂-¹⁵N₃]-guanosine at the known metal binding site, G10.1 and, for comparison, [2-¹³C-1,7,NH₂-¹⁵N₃]-guanosine at G16.2. The ¹⁵N NMR chemical shifts of the labeled N7s monitored during addition of Mg²⁺, Cd²⁺, and Zn²⁺ showed the same preference for binding at G10.1 over G16.2 for each metal. These results demonstrate that ¹⁵N labeling can be used to evaluate the binding of different metals, including Mg²⁺, to a given nitrogen, as well as to compare the binding potential of different sites.