Raman spectroscopic studies of the hepatitis delta virus (HDV) ribozyme

The general acid/base mechanism proposed for HDV catalysis involves a cytosine, C75 (genomic) / C76 (antigenomic), in proton transfer. Biochemical studies suggest that C75/C76 has a pK_a perturbed to near neutrality. In this thesis, the first objective is to directly measure the pK_a of C75 in a genomic HDV ribozyme using Raman crystallography. The results presented here reveal pK_a values for C75 that reflect anticooperative thermodynamic coupling with Mg²⁺ binding, with values of 6.15 and 6.40 in the presence of 20 and 2 mM Mg²⁺, respectively. These studies provide the first direct physical measurement of a pK_a near neutrality for a catalytic residue in a ribozyme.

The second objective is to investigate metal-RNA interactions in crystals of HDV ribozyme. Metal cation binding lies at the heart of much of RNA chemistry and is crucial for RNA folding and ribozyme catalysis. Here, we show Raman crystallography and Raman difference spectroscopy provide a unique means for probing metal-RNA interactions in HDV. The present studies focus on: (1) detecting the inner-sphere interactions between magnesium hydrate and the phosphate backbone of HDV by identifying and analyzing the vibrational signatures of inner-sphere coordinated magnesium hydrate (~322 cm^-1) and the PO₂^- symmetric stretch (~ 1100 cm^-1) perturbed by the formation of a PO₂^- —Mg²⁺ (H₂O)_x (x≤5) inner-sphere complex. About 5 inner-sphere Mg²⁺—^-O₂P contacts per HDV molecule are observed in the presence of 20 mM magnesium; (2) characterizing Mg²⁺—HDV and Co(NH₃)6³⁺—HDV interactions. Our results reveal that Mg²⁺ and Co(NH₃)63+ binding induce modest changes in HDV molecular conformation and Co(NH₃)₆³⁺ is able to displace some inner-sphere coordinated Mg²⁺ ions in HDV.

Finally, using Raman technique combined with ribozyme kinetic studies (the latter contributions are from the research group of Dr. B.Golden (Purdue University)), we observe an inner-sphere interaction of the catalytic Mg²⁺ ion with the N7 of guanine in the G1U wobble pair at the cleavage site. A new structural model for the active site of HDV is proposed based on the present results and previous studies. This model accommodates all available biochemical data on the HDV ribozyme.