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). (31)P NMR spectroscopy in conjunction with phosphorothioate substitutions is used in this study to investigate these putative metal sites. The (31)P 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 Cd(2+) to the hammerhead with an A9-S(Rp) or A9-S(S)(Rp) substitution results in a 2-3 ppm upfield shift of the (31)P NMR resonance. In contrast, the P1.1-S(Rp) and P1.1-S(Sp) (31)P NMR features shift slightly and in opposite directions, with a total change in delta of </=0.6 ppm with addition of up to 10 equiv of Cd(2+). No significant shifts are observed for an RNA.RNA duplex with a single, internal phosphorothioate modification upon addition of Cd(2+). Data obtained using model compounds including diethyl phosphate/thiophosphate, AMP, and AMPS, show that a Cd(2+)-S interaction yields an upfield shift for the (31)P NMR resonance, even in the case of a weak coordination such as with diethyl thiophosphate. Taken together, these data predict that Cd(2+) has a high affinity for the A9 site and suggest that there is flexibility in metal coordination within the binding pocket. Cd(2+) 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 (31)P NMR spectroscopy in conjunction with phosphorothioates as a probe for metal binding sites in nucleic acids.