Enzymatic structure mapping of 1/99 WT HDV. (A) Structure mapping of 1/99 WT monomer and dimer. Monomeric and dimeric RNAs were prepared for structure mapping as described in the Materials and Methods section “Enzymatic structure mapping of RNA;” purification gel not shown. Sequencing lanes for G (T1) and all nucleotides (OH−) are provided in the left portion of each data set. The secondary structures were determined using RNases A (C, U-specific), T1 (G-specific), T2 (single-stranded-specific), and V1 (double-strand-specific). Residues 1–15 were resolved on a separate gel, shown at the bottom. (B) Analytical native gel analysis of RNAs used for structure mapping. Gel is 10% native PAGE run in 0.5× TBE. This gel confirms that monomer and dimer identity have been retained upon purification. (C) Monomer cleavage data superimposed on the monomer secondary structure are consistent with published HDV ribozyme structure, and include the noncanonical N1-N1, carbonyl-amino base pair between the terminal A and G of P4 (Ferre-D'Amare et al. 1998). (D) Dimer cleavage data superimposed on an extended dimer secondary structure model. In C and D, secondary structures are mapped with RNases A (orange), T1 (blue), T2 (green), and V1 (red). Closed and open triangles represent stronger and weaker cleavages, respectively. Most notably, L4 is largely protected in the dimer.
