Lead Acetate Cleavage Assay for TectoRNA

TectoRNA assemblies were probed using a lead acetate cleavage assay as described previously (33 (link),38 (link),51 (link)). For each assembly, unlabeled tectoRNA at various concentrations ranging from 5 nM to 20 μM was mixed with ∼1 nM of 3′-[³²P]pCp labeled tectoRNA to monitor assembly and cleavage. For the cleavage assays, tectoRNA assemblies were first denatured at 95°C for 1 min and immediately cooled on ice for 2 min. Using a thermocycler, each tectoRNA sample was incubated at 20°C for 5 min before the addition of association buffer containing 25 mM HEPES pH 7.5, 50 mM KOAc, and either 0.05 mM, 0.5 mM or 2 mM Mg(OAc)₂ in the presence or absence of 200 μM folinic acid (FA). Following a 30-min incubation period at 20°C, 1 μl of 10 mg/ml tRNA was added prior to addition of 10 mM (final) Pb(OAc)₂. After 5 min at 20°C, the reaction was quenched with 10 μl of 100 mM EDTA solution. Each sample was ethanol precipitated, washed, and resuspended in colorless loading buffer (10 M urea, 1.5 mM EDTA) prior to loading onto a 10% (19:1) denaturing PAGE gel. Labeled RNA molecules were visualized using a phosphor-screen and a Typhoon Biomolecular Imager (Amersham). Lane profiles and densitometry analysis were analyzed using ImageJ 2.0 software.