Mn2+-nitrogen interactions in RNA probed by electron spin-echo envelope modulation spectroscopy: Application to the hammerhead ribozyme

We report application of electron spin-echo envelope modulation (ESEEM) spe ctroscopy to the problem of metal coordination environments in structured R NA molecules. ESEEM has been used in conjunction with N-15-guanosine labeli ng to identify nitrogen ligation to a Mn2+ site in a hammerhead ribozyme an d in Mn2+-model guanosine monophosphate (GMP) complexes. Hammerhead ribozym e complexes consisting of a 34-nucleotide RNA enzyme strand annealed to a 1 3-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-affin ity Mn2+ site (Horton, T. E.; Clardy, R. D.; DeRose, V. J. Biochemistry 199 8, 51, 18094-18108). Significant modulation of the electron spin-echo from several low-frequency features is detected for the natural-abundance, N-14- hammerhead samples. At 3600 G, the main hammerhead three-pulse ESEEM featur es 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 N-15-guanosine incorporated into the enzyme strand, as well as for an N-15-labeled Mn2+-G MP complex, the modulation is completely altered and consists of one main f eature at 3.4 MHz and a smaller feature at the upsilon(n)(N-15) Larmor freq uency of 1.6 MHz. Preliminary analysis of the ESEEM data reveals an apparen t hyperfine coupling of A(N-14) similar to 2.3 MHz, similar to previously r eported values for Mn2+ directly coordinated to histidine and imidazole. Th ese data demonstrate the potential for ESEEM as a spectroscopic tool for me tal ligand determination in structured RNA molecules.