Figure 1. Strong experimental and computational evidence for a conformational capture binding mechanism by HIV‐1 TAR ligands [ 22]. (a) Constitution of the ligands and representative structures of the RNA–ligand complexes circling the structure of free TAR RNA and its secondary structure. Ligand names, Protein Data Bank (PDB) codes, and the average angle of the interhelical bending are given. (b) Overlay of the ligand‐bound structures (gray, PDB codes lie adjacent) and three selected conformers (green, subconformers in light green), aligned along the terminal helix I.

Figure 2. NMR spectra of the guanine‐sensing riboswitch aptamer domain (a) – HSQC overlay of ligand‐free (red) and ligand‐bound (black) RNA with imino proton resonance assignment of RNA–ligand complex [ 45] (‐labeled RNA, unlabeled ligand hypoxanthine, 600 MHz, 283 K); (inset) secondary structure of ligand‐bound state with gray solid lines: Watson–Crick base‐pairing interactions; gray dashed lines: noncanonical base‐pairing interactions (construct numbering according to Breaker et al. [ 54]). (b) One‐dimensional imino proton NMR spectra of ligand‐free RNA (red) and RNA–ligand complex (black). NMR imino proton resonances (H1 and H9) of the complexed ligand hypoxanthine are annotated.

Figure 3. (a) Sequence and secondary structure of the temperature‐sensitive hairpin 2 of the Salmonella 4U RNA thermometer (4U‐hairpin2). The 4U motif (gray) and the opposing Shine–Dalgarno (orange) sequence are highlighted. (b) CSP of the – imino group resonances of the nucleobases U12 (colored blue in the secondary sequence) of the 4U‐hairpin2 RNA plotted against the [Mg ²⁺]/[RNA] ratio and fitted according to Equation 3 (straight line). (c) Mg ²⁺ binding model assuming two distinct Mg ²⁺ binding sites on the RNA. The RNA can adopt four different states (I, II, III, IV). Transitions between these states are described by microscopic dissociation constants (k ₁, k ₂, k ₃, k ₄). Transitions between free RNA, binary, and ternary complex are described by macroscopic dissociation constants (K ₁, K ₂). Equations describing interdependencies of the different dissociation constants are also given. K ₁, K ₂ as well as k ₁, k ₂, k ₃, k ₄ are thermodynamic constants that describe the RNA–[Mg ²⁺] _n complexes under equilibrium conditions.

Figure 4. (a) Sequence and secondary structure of the HIV type 2 TAR RNA (HIV‐2 TAR). (b) Configuration of the tripeptide ligand with two arginine residues flanking a synthesized, 2‐pyrimidinyl‐bearing amino acid (Arg‐pyrimidinyl‐Arg‐NH ₂). (c) one‐dimensional NMR spectra of the imino proton region of HIV‐2 TAR with increasing amounts of Arg‐pyrimidinyl‐Arg‐NH ₂ as ligand. The RNA/ligand ratios are specified at the right end of the spectra. Selected resonances are assigned. (d) Plots of the CSPs against the ratio of ligand and RNA concentration of selected imino protons that illustrate the different behaviors. G26 (red) represents a residue that is only effected at lower ratios, while U42 (green) changes its shifting direction at a ratio of about 2–3 : 1. At this ratio G17 (blue) begins to shift. (e) Sections of the two‐dimensional – TOCSY spectra of HIV‐2 TAR overlaid with increasing amounts of ligand. The section shows the cross‐peaks between the pyrimidine protons H5 and H6. Significant changes in the CSPs can be observed at ligand/RNA ratios of 2–3 : 1.

Figure 5. PRE of the imino resonances of the 4U‐hairpin2 RNA observed upon the addition of 4 µM MnCl ₂. Changes in linewidths (Δν) (Hz) in the dimension are shown with the corresponding scale on the left side of the diagram (red triangles) and changes in intensity (ln(I[0 µM]/I[4 µM]) (a.u.)) are shown with the corresponding scale on the right (blue squares). U32 and U31 do not give rise to any observable imino signals, and the imino signals of G30 and U4 are too weak to be analyzed.

Figure 6. (a) Two‐dimensional – NOESY spectrum of the 27‐nucleotide neomycin riboswitch RNA in complex with the aminoglycoside antibiotic ribostamycin (insert) in H ₂O; section showing NOE cross‐peaks between the RNA imino protons and ribostamycin resonances in the region between 3.5 and 0.5 ppm, which is free of RNA resonances. RNA resonances involved in the binding site (highlighted in red) show strong NOEs to the ligand. (b) Section of a two‐dimensional ‐filtered/‐edited – NOESY in D ₂O selectively monitoring intermolecular NOE contacts between ribostamycin ( ¹²C‐bound) and RNA (‐bound) protons. RNA assignments are given in black, ribostamycin assignments in red. A residual intramolecular RNA NOE is labeled by an asterisk. Intense NOE contacts exist between the H2 of the A17 and various ligand protons (gray bar). This loop nucleotide is looped out and lies as a flap on top of the ligand in the RNA–ligand complex. (c) Secondary structure of the bound RNA, localization of the ligand‐binding pocket, and rough orientation of the ligand based on NOE data. The ligand and the RNA regions in close contact to it are color coded accordingly.

Figure 7. Section from the two‐dimensional – NOESY experiment performed on a 0.9 mM 4U‐hairpin2 RNA sample in the presence of 5 mM [(Co(NH ₃) ₆] ³⁺. Assignments of the cross‐signals between protons of the [Co(NH ₃) ₆] ³⁺ and the imino protons of the RNA are given. On top of the NOESY spectrum, the corresponding imino region of the one‐dimensional ‐NMR spectrum is given. Buffer conditions: 15 mM K _xH _yPO ₄, pH 6.5, 25 mM KCl, 90% H ₂O, and 10% D ₂O.