Mechanistic Analysis of the Hepatitis Delta Virus (HDV) Ribozyme: Methods for RNA Preparation, Structure Mapping, Solvent Isotope Effects, and Co-transcriptional Cleavage

Abstract

Small ribozymes such as the hairpin, hammerhead, VS, glm S, and hepatitis delta virus (HDV) are self-cleaving RNAs that are typically characterized by kinetics and structural methods. Working with these RNAs requires attention to numerous experimental details. In this chapter we focus on four different experimental aspects of ribozyme studies: preparing the RNA, mapping its structure with reverse transcription and end-labeled techniques, solvent isotope experiments, and co-transcriptional cleavage assays. Although the focus of these methods is the HDV ribozyme, the methods should be applicable to other ribozymes.

Inverse Thio Effects in the Hepatitis Delta Virus Ribozyme Reveal that the Reaction Pathway Is Controlled by Metal Ion Charge Density

“…The final metal chloride solutions’ concentrations (1×) were variable and ranged from 250 mM to 4 M. A detailed description of the preparation of the buffers is described in our earlier studies. 34 , 38 The buffers were brought to the appropriate pH with HCl for low pH buffers or the hydroxide of the cation under study (i.e., NaOH, KOH, LiOH, or NH 4 OH) for high pH buffers. The same buffers were also prepared in D 2 O for the proton inventory experiments, details of which are described below.…”

“…Proton inventory experiments were performed in the presence of Li + , Ba 2+ , or NH 4 + at pL= 5.4, as described previously. 33 , 34 , 38 All the buffers, metal ion mixtures, and RNA were prepared in either 100% H 2 O or 100% D 2 O. The pD was calculated according to eq 4 : 39 …”

“…Calculations of the mole fraction took into account differences in the density of the two solvents, as described. 33 , 34 , 38 …”

Determination of hepatitis delta virus ribozyme N(–1) nucleobase and functional group specificity using internal competition kinetics

“…The wild-type HDV ribozyme and site-specific mutant ribozymes were prepared by T7 RNA polymerase transcription from cloned plasmid DNA templates (Table S1) containing the required promoter sequence using standard procedures [28]. The trans-cleaving version of the HDV ribozyme used in these studies is derived from the antigenomic sequence and contains a shortened P4 stem and a discontinuous J1/2 region (see Fig.…”

Rapid Kinetics of Pistol Ribozyme: Insights into Limits to RNA Catalysis

“…The pH meter was calibrated at 25 °C and readings for buffers and solutions were measured at 25 °C; for solutions using D 2 O, the pD was determined by adding 0.4 to the pH reading. The effect of 2 M NaCl on pH meter response using the calibration curve for Na + -containing solutions from Chadalavada et al Stopped-flow kinetics experiments for measurement of D 2 O effects were performed essentially as described, above. The observed rate constants were plotted as a function of pH or pD accordingly and fit to a general equation for two ionizable active site groups acting as acid and base …”