Cloning and Expression of TBP6.7 β2-β3 Loop Peptide

The β2–β3-loop peptide sequence of TBP6.7 (Figure 4A) was cloned into the pB33eCPX construct (41 (link)) (AddGene) using restriction enzymes NdeI and XhoI (NEB), downstream of an in-frame myc tag and transformed into 5-alpha competent E. coli cells (NEB). The eCPX-β2–β3-loop plasmid DNA was purified by miniprep (Omega) and ∼200 ng were used to electroporate E. coli MC1061 F⁻ cells (Lucigen) in 1 mm electroporation cuvettes (Fisher). Cells were grown in 50 ml LB (Fisher) containing 12.5 μg ml⁻¹ chloramphenicol (GoldBio Technology) at 37 °C to an OD₆₀₀ of 0.5 and induced overnight with 0.1% arabinose at 25 °C. ∼5 × 10⁸ cells were pelleted (7300 × g) for 5 min at 4 °C, then washed with ice-cold CellGro PBS 1× (Corning). Cells were incubated with 100 nM Cy5-labeled TAR RNA (IDT) (annealed by heating at 95 °C for 2 min, followed by plunging into ice) and 1:1000-fold diluted FITC-conjugated anti-cMyc antibody (Abcam) with rotating at 4 °C in 1 ml PBS for 1 h. Cells were pelleted and washed once with ice-cold PBS. RNA-binding (Cy5 fluorescence) and display (FITC fluorescence) were measured using a CyAn ADP flow cytometer (Beckman-Coulter). All flow data were analyzed and plotted using FlowJo 10.3.