Figure 3

3× pen aRNA cleavage by Pen17zyme1B and Pen17zyme2. Cleavage reaction products were analyzed on denaturing 8% PAGE. Incubation of 3×pen aRNA (160 nt) with the Pen17zyme1B and Pen17zyme2 resulted in accumulation of 71-nt final excision product and 137-nt semi-product. (A) Lane 1, total RNA from E. coli JM109(DE3)/pCP3×3 enriched for low molecular weight RNAs (total RNA); Lane 2, low molecular weight DNA ladder (LWM); Lane 3, 3×pen aRNA; Lanes 4-6, Cleavage products of 3×pen aRNA after 17 hours of incubation with 3×pen aRNA: Pen17zyme1B: Pen17zyme2 molar ratio 1:1:1, 1:10:1, and 1:10:2, respectively. (B) Lane 1, total RNA; Lane 2, LWM; Lane 3, 3×pen aRNA; Lane 4, Cleavage products of 3×pen aRNA after 40 hours of incubation with 3×pen aRNA: Pen17zyme1B: Pen17zyme2 molar ratio 1:10:1. (C): Lane 1, total RNA; Lane 2, LWM; Lane 3, 3×pen aRNA; Lane 4, Cleavage products of 3×pen aRNA after 40 hours of incubation with 3×pen aRNA: Pen17zyme1B: Pen17zyme2 molar ratio 1:10:10. (D): Lane 1, LWM; Lane 2, total RNA; Lane 3, equimolar mixture of Pen17zyme1B and Pen17zyme2; Lane 4, 3×pen aRNA; Lane 5, Cleavage products of 3×pen aRNA after 72 hours of incubation with 3×pen aRNA: Pen17zyme1B: Pen17zyme2 molar ratio 1:10:10. Cleavage reactions were performed under conditions described in legend to Figure 2 except for the incubation time and 3×pen aRNA: DNazymes molar ratio, which were as indicated above.