Figure 2

3× pen aRNA cleavage by Pen17zyme1, Pen17zyme1B or Pen17zyme1C. Cleavage reaction products were analyzed on denaturing 8% PAGE. The expected cleavage products are 94-nt 5' terminal and 66-nt 3' terminal halves of 160-nt 3×pen aRNA. Lane 1, 3×pen aRNA cleavage with Pen17zyme1; Lane 2, 3×pen aRNA cleavage with Pen17zyme1B; Lane 3, 3×pen aRNA cleavage with Pen17zyme 1C. Cleavage reactions were set up as follows: RNA substrate and indicated DNAzyme were annealed in 50 mM MOPS-NaOH (pH 7.2), 500 μM spermine by incubating the mixture at 90°C for 2 min followed by cooling to 23°C within 10 min. After annealing, the composition of the mixture was adjusted to 125 mM KCl, 500 mM NaCl, 7.5 mM MgCl₂, 15 mM MnCl₂, 150 μM spermine, and 50 mM MOPS-NaOH (pH 7.2), with 3×pen aRNA: DNAzyme molar ratio 1: 5. The reaction was performed at 23°C for 16 hours, and stopped by ethanol precipitation of the products.