Fig. 2: Force spectroscopy of SARS-CoV-2 pseudoknot.
a Schematic of force spectroscopy assay: a single RNA pseudoknot is tethered by duplex handles attached to beads held in optical traps. Pseudoknot secondary structure color-coded as in Fig. 1. b Unfolding force-extension curves (FECs) with 6-nt spacer between handle and pseudoknot (inset: schematic of conformers N and N′ showing handle location). Black: FECs showing full-length unfolding of pseudoknot without intermediates. Magenta: FECs unfolding pseudoknot via intermediate state I1. Green: FECs showing unfolding of alternate stem-loop structures (labeled Alt). Dashed lines: WLC fits to each state (orange: N/N′, cyan: I1, gray: U, brown: Alt; navy: intermediate for Alt). FECs offset for clarity. c FECs showing full-length refolding of pseudoknot, with intermediates corresponding to folding of stem 1 (Ir1, cyan dashed line) and stem 1 + stem 3 (Ir2, purple dashed line). d Unfolding force distribution for pseudoknot (N = 745 FECs from 10 molecules) shows two peaks, representing the distinct conformers N and N′. Blue: fit to single-population landscape model; red: fit to two-population landscape model.
