Metal−Phosphate Interactions in the Hammerhead Ribozyme Observed by 31P NMR and Phosphorothioate Substitutions

Abstract

The hammerhead ribozyme is a catalytic RNA that requires divalent metal cations for activity under moderate ionic strength. Two important sites that are proposed to bind metal ions in the hammerhead ribozyme are the A9/G10.1 site, located at the junction between stem II and the conserved core, and the scissile phosphate (P1.1). ³¹P NMR spectroscopy in conjunction with phosphorothioate substitutions is used in this study to investigate these putative metal sites. The ³¹P NMR feature of a phosphorothioate appears in a unique spectral window and can be monitored for changes upon addition of metals. Addition of 1−2 equiv of Cd²⁺ to the hammerhead with an A9-S_Rp or A9-S_Sp substitution results in a 2−3 ppm upfield shift of the ³¹P NMR resonance. In contrast, the P1.1-S_Rp and P1.1-S_Sp³¹P NMR features shift slightly and in opposite directions, with a total change in δ of ≤0.6 ppm with addition of up to 10 equiv of Cd²⁺. No significant shifts are observed for an RNA·RNA duplex with a single, internal phosphorothioate modification upon addition of Cd²⁺. Data obtained using model compounds including diethyl phosphate/thiophosphate, AMP, and AMPS, show that a Cd²⁺−S interaction yields an upfield shift for the ³¹P NMR resonance, even in the case of a weak coordination such as with diethyl thiophosphate. Taken together, these data predict that Cd²⁺ has a high affinity for the A9 site and suggest that there is flexibility in metal coordination within the binding pocket. Cd²⁺ interactions with the cleavage site P1.1-S positions are weaker and appear to be stereospecific. These data have implications for mechanisms that have been proposed to explain the influence of metal ions on hammerhead ribozyme activity. These experiments also show the potential utility of ³¹P NMR spectroscopy in conjunction with phosphorothioates as a probe for metal binding sites in nucleic acids.