Synthesis and Expression of Chimeric Proteins

BG4,^{31 (link)} conjugated with FluoProbes647H (Cy5-BG4), was obtained from Absolute Antibody (product number Ab00174-1.1). TMPyP4 was obtained from Sigma-Aldrich (catalog number 613560). N-terminal glutathione S-transferase (GST) tagged human nucleolin IGF2, CNBP, and helicase plasmids were synthesized by GenScript. Purified, recombinant bovine DHX36^{41 (link)} was provided as a gracious gift by the Ferré-D’Amaré Lab (National Institutes of Health, Bethesda). The sequences of all proteins used are listed in Table S1. All chimeric proteins were expressed via in vitro translation (IVT) reactions using the PURExpress In Vitro Protein Synthesis Kit (NEB) as described previously.^{23 (link)} For all IVT reactions, 288 ng of plasmid was added to 80 μL of a IVT mixture, and reactions were carried out at 37 °C for 2 h. Expression of all protein constructs was confirmed via Western blot (Figure S2).