Visualization of intermediates and D-RNA hydrolysis with L-hammerhead ribozyme (L-HH)
D-RNA substrate of 14 nucleotide long (0.25 μM) labelled with FAM was incubated with 2.5 μM L-HH ribozyme labelled with cy5 and 4 μM cold L-HH at 70°C during 2 min, then mixture was fast cooled down, then reaction was incubated during 3h at 37°C in presence of increasing concentration of MgCl2 (0.5, 1, 5, 10, 15, 20, 25 mM, lanes 3, 4, 5, 6, 7, 8, 9, respectively) in binding buffer (50 mM Tris-HCl pH 7.5, 160 mM KCl, 10 mM spermidine). Analysis was done on 15% native polyacrylamide gel with 2.5% glycerol. Lane: 1- D-RNA; 2 – L-HH ribozyme.