Figure 4.

(A) Scheme for interference rescue. Ribozyme activity is reduced by mutating a G that is intolerant of IMPαS substitution to an A. Activity is rescued by substituting the A with DMPαS. (B) A portion of the sequencing autoradiogram showing the reaction of L-21 G⁴¹⁴ G303A DMPαS or AMPαS with rT(-1)S for 10 min at 50°C in 4 mM MgCl₂. The nucleotide positions correlating to the cleavage products are shown to the left for lanes 1–4 and to the right for lanes 5 and 6. For the 5′ control (lanes 5 and 6), the L-21 G⁴¹⁴ RNAs are enzymatically labeled at the 5′ end. This results in a reversal of the sequence orientation on the gel, but shows the actual level of analog incorporation at all nucleotide positions. A visual comparison between the reaction and the 5′ control is complicated by strong phosphorothioate interference at A302 and A306. These bands are absent in both the DMPαS and AMPαS reaction lanes, but they are present in both 5′ controls.