Figure 3.

The effect of varying the concentration of MgCl₂ on cleavage efficiency. Synthetic s1 RNA (174 nts long) (A, lane 2 and B, lane 1) was used for the cleavage reaction in equimolar ratio (0.3 pmols) with the two Rzs separately. (A) The results obtained with Rz-553 are shown. Lane 1 depicts the synthesis of Rz-553 as described before. Lane 2 depicts the labeled s1 synthetic transcript that is 174 bases long. No cleavage was observed if the MgCl₂ was omitted from the reaction (lane 3) or when it was present at a concentration of 0.1 mM (lane 4). Specific cleavage could be observed at 1 mM MgCl₂ (lane 5). The cleavage efficiency increased significantly in the presence of increasing amounts of MgCl₂ (lane 6, 3 mM; lane 7, 5 mM; lane 8, 10 mM; lane 9, 25 mM; and lane 10, 50 mM). (B) Lane 1 is same as lane 2 of A. No cleavage was observed in the absence of MgCl₂ (lane 2) when equimolar amounts of Rz-984 were used. Lane 3 shows the extent of cleavage in the presence of 2 mM MgCl₂. Specific cleavage products were also obtained under simulated physiological conditions (150 mM KCl, pH 7.5, 37°C, 2 mM MgCl₂) (lane 4). The cleavage efficiency did not change in the presence of 5 mM (lane 5) or 10 mM MgCl₂ (lane 6). Increased cleavage was, however, observed in the presence of 50 mM MgCl₂. (C) A comparison of the cleavage efficiency of the two Rzs at varying concentration of MgCl₂ is shown. It is evident from the graph that more than 40% cleavage of the target RNA is achieved by Rz-553 in presence of 5 mM MgCl₂ as compared with only 20% with Rz-984 under identical experimental conditions. Also note that even in the presence of 50 mM MgCl₂ the cleavage reaction did not proceed to completion.