Decade markers system

If not indicated otherwise, 6 pmol template 3′ RNA were incubated with 4 pmol L protein and NTPs (0.2 mM UTP/ATP/CTP and 0.1 mM GTP supplemented with 166 nM 5 μCi [α]³²P-GTP) in 10 μL polymerase reaction buffer (100 mM HEPES(NaOH) pH 7.0, 150 mM NaCl, 50 mM KCl, 1 mM MnCl₂, 1% (w/w) glycerol, and 2 mM dithiothreitol). The samples were incubated at 30°C for 1 h. The reaction was stopped via the addition of an equivalent volume of RNA loading dye (98% formamide, 18 mM EDTA, 0.025 mM SDS, xylene cyanole and bromophenol blue) and heating the samples at 95°C for 3 min. Products were separated by gel electrophoresis on denaturing 7 M urea, 20% polyacrylamide Tris–borate–EDTA gels in 0.5-fold Tris–borate buffer. The Decade markers system (Ambion) was used as molecular weight marker. Signals were detected via phosphor screen autoradiography using a Typhoon scanner (GE Healthcare).
In samples containing 5′ RNA, the 5′ RNA was incubated with L protein for 15 minutes on ice to allow protein-RNA complex formation prior to the addition of template RNA and NTPs.
For primer elongation assays, 6 pmol template RNA were incubated with a tenfold excess of primers for 15 minutes on ice prior to the addition of L protein and NTPs to allow for primer annealing.

Synthetic vRNA loop (‘v51_mut_S’) (5′-pAGU AGA AAC AAG GGU GUA UUU UCC CCU CUU UUU GUU UCC CCU GCU UUU GCU -3′) (IDT) was used for all in vitro replication activity assays. For all de novo replication activity assays, 2.4 μM FluPol Zhejiang-H7N9 (WT or 4 M) were mixed with (i) 0.8 μM v51_mut_S and/or (ii) 8 µM ANP32A and/or (iii) 16 µM pS5 CTD(6mer) (respective molar ratio: 3 FluPols: 1 template: 10 ANP32: 20 pS5 CTD). Reactions were launched at 30 °C for 4 h by adding ATP/GTP/CTP/UTP (AGCU) or only ATP/GTP/CTP (AGC), α-32P ATP (PerkinElmer) and MgCl₂ in a final assay buffer containing 50 mM HEPES pH 8, 150 mM NaCl, 2 mM TCEP, 100 μM/NTP, 1 mM MgCl₂, 0.05 μCi/μl α-32P ATP. Reactions were stopped by adding 2X RNA loading dye, heating 5 min at 95 °C and immediately loaded on a 20% TBE-7M urea-polyacrylamide gel. Each gel was exposed on a storage phosphor screen and read with an Amersham Typhoon scanner (Cytiva). For each gel the decade markers system (Ambion) was used.