NMR characterization and ligand binding site of the stem–loop 2 motif from the Delta variant of SARS-CoV-2

(A) Determination of estimated K_D via ligand-detected ¹H-1D NMR titration. (B) RNA-based Euclidean distance plot to determine the binding site of P6E06 to s2m Delta. The structural formula of the ligand is shown in the middle. Internal loop residues are highlighted in green and loop residues are highlighted in magenta. A threshold of twofold the standard deviation was used. (C) ¹H, ¹³C HSQC spectra of the aromatic region of s2m (100 µM) with 0 µM (black) and 500 µM (yellow) P6E06. Resonance assignments are annotated and the notation of nucleotides corresponds to the position in the genome −29,000. Loop resonances are labeled in magenta and internal loop resonances are labeled in green. (D) The secondary structure of s2m Delta is colored with HSQC-based CSP heatmap (strong: red, weak: blue), and red corresponds to the maximum CSP detected.