RNAP Crosslinking Assay Protocol

CPR crosslinking assays (Figure 5) were performed as described previously (Nayak et al., 2013 (link)). Nucleic acid scaffolds were prepared by annealing RNA, template DNA (T-DNA) and 15 μM non-template DNA (NT-DNA) at 10 µM, 12 µM, and 15 µM final concentrations, respectively, in reconstitution buffer (RB; 20 mM Tris-HCl pH 8, 20 mM NaCl, and 1 mM EDTA). ECs, ePECs, and his PEC were formed by incubating 1 μM RNAP and scaffold (2 μM, based on RNA) in buffer A (50 mM Tris-HCl pH 8, 20 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, and 2.5 ug acetylated bovine serum albumin/mL for 15 min at room temperature (RT). For crosslinking reactions with NTP, 3′deoxyECs formed by reaction with 3′dNTP were incubated for 15 min at RT with 0, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, and 10 mM GTP or ATP. Next, EC, ePEC, or his-PEC (final RNAP 0.8 uM and scaffold 1.6 uM) were incubated for 60 min with 2.5 mM CSSC and 0.05 mM DTT (E = –0.16) and stopped with 50 mM iodoacetamide. Samples were separated by native PAGE to verify reconstitution efficiency and by sodium dodecyl sulfate (SDS)-PAGE using 4–15% GE Healthcare PhastGel to quantify formation of crosslinks. Gels were stained with Coomassie Blue and imaged with a CCD camera. The fraction cross-linked was quantified with ImageJ software.