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.