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 ν n (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.

Metal ions are critical to the structure and function of many RNA molecules, but measuring detailed coordination environments in RNA is challenging under solution conditions. The phosphodiester bond cleavage reaction of the hammerhead ribozyme is activated by Mn 2+ , which provides a paramagnetic probe for EPRbased spectroscopic techniques. In this study, EPR and continuous-wave Q-band (34 GHz) electron nuclear double-resonance (ENDOR) spectroscopies have been used to investigate the coordination environment of a high-affinity Mn 2+ site in the hammerhead ribozyme. Small changes in low-temperature X-band EPR signals are detected as signatures of Mn 2+ ions in the RNA binding pocket. 1 H and 31 P Q-band ENDOR spectra are presented for Mn-hammerhead and Mn-nucleotide model complexes. The 31 P ENDOR data allow discrimination between direct Mn 2+ -phosphodiester coordination versus coordination through a hydrogenbonded water molecule. Observation of hyperfine-coupled 31 P (A( 31 P) ∼4 MHz) provides evidence for direct coordination to a phosphodiester group in the hammerhead Mn 2+ site. Exchangeable protons from aqueous ligands and nonexchangeable protons from base ligands also are examined for the Mn-nucleotide and Mnribozyme complexes. These signals indicate an ordered site for Mn 2+ in the hammerhead ribozyme and allow the ligand environment to be predicted, demonstrating the potential of ENDOR spectroscopy as a probe of RNA-metal interactions.

Therapeutic study, level IV.

This retrospective review of a methicillin-resistant Staphylococcus aureus decolonization protocol using CHG bath/shower and PI-SNA nasal painting revealed a significant decrease in the infection rate of patients undergoing lower extremity fracture repairs. We recommend its use without contraindications, but recognize that additional investigations are necessary.

No abstract

No abstract

No abstract

Therapeutic study, level IV.