G50 spin column

In a first step, actin pre-mRNA was prepared by transcription in vitro with T7 RNA polymerase (S1 Text). The transcription reaction was not gel-purified, but instead was precipitated with ethanol. After washing twice with 70% ethanol, the precipitated RNA was dried, dissolved in 50μl CE buffer (10 mM cacodylic acid-KOH, pH 7.0, 0.2 mM EDTA-KOH, pH 8) or water and applied to a G 50 spin column (GE Healthcare). The eluted RNA was then subjected to DNA enzyme cleavage, essentially as described previously [25 (link)]. First, a threefold molar excess of the DNA enzyme over the pre-mRNA was added to the reaction mixture. The solution was then adjusted to 15 mM NaCl and 5 mM TRIS-HCl, pH 7.7. After denaturation at 70°C for 2 min the mixture was kept at room temperature for 5 min. Finally, 150 mM NaCl, 50 mM Tris-HCl, pH 7.7 and 2 mM of both MgCl₂ and MnCl₂ were added and the mixture was incubated at 30°C for 3 hrs. To remove the cyclic phosphate produced at the 3’ end of the 5’ fragment by the DNA enzyme, the intrinsic 3'-phosphatase activity at low ATP concentration of T4 polynucleotide kinase (T4 PNK) was used [26 (link)]. The reaction was supplemented with 2 unites/μl T4 PNK, PNK buffer and 0.4 mM ATP and incubated for 1 h at 37°C [26 (link)]. The RNA digestion fragments were gel-purified as described for in vitro transcriptions (S1 Text).

Cell fractionation and extraction of crude RSV and PIV-1 RNP complexes were performed as described [23 (link)]. Unless otherwise specified, nucleoside triphosphate (NTP) concentrations were around their K_m value: 0.1 μM CTP, 1 μM ATP, 2 μM UTP, and 500 μM GTP. The radioactive tracer was either 10 μCi [α-³³P]rCTP (CTP competitive mode) or 15 μCi [α-³³P]rGTP (non-CTP competitive mode) together with 0.1 μM GTP. The NTP concentration for the competition assay was either 1 or 100 μM for CTP, ATP, GTP, and UTP. The reaction was initiated with the addition of 1.5 μL of the virus-infected cell extract and incubated for 2 hours at 30°C in a final volume of 30 μL. Unless otherwise specified, the standard reaction was stopped by adding 25 μL of Tris-sodium-EDTA buffer containing 10 mM Tris-HCl pH 8, 150 mM NaCl, and 100 mM EDTA. Processing of samples included elution through a G-50 spin column (GE Healthcare), phenol-chloroform extraction, and ethanol precipitation. Air-dried RNA samples were reconstituted in 20 μL TBE urea gel loading buffer (Invitrogen), and migrated through a 6% TBE urea PAGE gel for 1 hour at 190 V. The gel was dried and exposed to a phosphor-screen that was scanned with a Storm 860 phosphorImager (Molecular Dynamics). Biochemical assays for the inhibition of influenza and HCV polymerases were performed as previously described [17 (link),27 (link)].