Phosphor imaging

Radiolabeled crRNA repeat substrates were generated by in vitro transcription as described previously ((Carte et al., 2008 (link)), see Table S1 for oligo sequences). RNA binding and cleavage assays were carried out as described previously (Carte et al., 2008 (link)) with the following modifications. Sth Cas6 was incubated with 5,000 cpm of uniformly ³²P-labeled RNA substrate in 25 mM Tris-HCl (pH 7.0), 0.75 mM DTT, 1.5 mM MgCl₂, 5 μg E. coli tRNA, and 10% glycerol. The reaction conditions for Sth Cse3 (Cas6e) were identical except that DTT was replaced with 5 mM β-mercaptoethanol and NaCl was added to 50 mM. Reactions were incubated at 37° C for 30 minutes prior to electrophoretic separation on both native TBE 8% polyacrylamide (RNA binding) and 7M urea TBE 15% acrylamide (RNA cleavage) gels. The gels were dried and RNAs detected by phosphor imaging (Amersham).

A 3-fold excess of PPT-57 DNA template was heat-annealed to 50 nM 5′-fluorolabeled PPT-17 primer, then incubated with 250 nM of RT in a buffer containing 50 mM Tris-HCl (Sigma, St. Louis, MO), pH 7.8, 50 mM NaCl (Sigma), 0.3 mM EDTA (Sigma), and 0.5 μM of each of dATP, dTTP, dGTP, and dCTP (GE Healthcare, Pittsburgh, PA) (33 (link)). The samples were preincubated at 37°C for 5 minutes before starting the reactions. For inhibitor dose-response experiments, each of the four inhibitors was titrated up to 100 μM, and the reaction was initiated with 6 mM MgCl₂ and allowed to proceed for 3 minutes. The reaction was stopped with 100% formamide loading dye containing traces of bromophenol blue. Samples were resolved on a 12% denaturing polyacrylamide gel followed by phosphorimaging (Amersham Biosciences, Piscataway, NJ). For the dose-response experiments, pausing sites caused by inhibition were quantified and summed; the % inhibition was calculated as the total amount of inhibited product divided by the amount of full-length product plus inhibition products, multiplied by 100. The product fractions were normalized and plotted against inhibitor concentration using GraphPad Prism software; the normalized data was fitted to a log[inhibitor] versus response curve with variable slope to extract IC₅₀ values for the inhibition of the RT enzymes by the α-CNP.