Abstract:

<p>The DIR2s RNA aptamer, a second-generation, in-vitro selected binder to dimethylindole red (DIR), activates the fluorescence of cyanine dyes, DIR and oxazole thiazole blue (OTB), allowing detection of two well-resolved emission colors. Using Fab BL3-6 and its cognate hairpin as a crystallization module, we solved the crystal structures of both the apo and OTB-SO bound forms of DIR2s at 2.0&thinsp;Å and 1.8&thinsp;Å resolution, respectively. DIR2s adopts a compact, tuning fork-like architecture comprised of a helix and two short stem-loops oriented in parallel to create the ligand binding site through tertiary interactions. The OTB-SO fluorophore binds in a planar conformation to a claw-like structure formed by a purine base-triple, which provides a stacking platform for OTB-SO and an unpaired nucleotide, which partially caps the binding site from the top. The absence of a G-quartet or base tetrad makes the DIR2s aptamer unique among fluorogenic RNAs with known 3D structure.</p>