Mn²⁺−Nitrogen Interactions in RNA Probed by Electron Spin−Echo Envelope Modulation Spectroscopy: Application to the Hammerhead Ribozyme

Abstract

We report application of electron spin−echo envelope modulation (ESEEM) spectroscopy to the problem of metal coordination environments in structured RNA molecules. ESEEM has been used in conjunction with 15N-guanosine labeling to identify nitrogen ligation to a Mn2+ site in a hammerhead ribozyme and in Mn2+−model guanosine monophosphate (GMP) complexes. Hammerhead ribozyme complexes consisting of a 34-nucleotide RNA enzyme strand annealed to a 13-nucleotide DNA substrate strand were poised in 1 M NaCl as a 1:1 complex with Mn2+, conditions previously determined to populate a single high-affinity Mn2+ site (Horton, T. E.; Clardy, R. D.; DeRose, V. J. Biochemistry 1998, 51, 18094−18108). Significant modulation of the electron spin−echo from several low-frequency features is detected for the natural-abundance, 14N-hammerhead samples. At 3600 G, the main hammerhead three-pulse ESEEM features arise at 0.6, 1.9, 2.5, and 5.2 MHz and are nearly identical for a Mn2+−GMP complex under the same conditions. For a ribozyme having 15N-guanosine incorporated into the enzyme strand, as well as for an 15N-labeled Mn2+−GMP complex, the modulation is completely altered and consists of one main feature at 3.4 MHz and a smaller feature at the νn(15N) Larmor frequency of 1.6 MHz. Preliminary analysis of the ESEEM data reveals an apparent hyperfine coupling of A(14N) ∼ 2.3 MHz, similar to previously reported values for Mn2+ directly coordinated to histidine and imidazole. These data demonstrate the potential for ESEEM as a spectroscopic tool for metal ligand determination in structured RNA molecules.