Exploring the energetic and conformational properties of the sequence space connecting naturally occurring RNA tetraloop receptor motifs

Thermodynamic fingerprints provide information about the sequence-conformation landscape. (A) Schematic of how differences in TL • TLR-binding geometry and scaffold geometry lead to conformational penalties in a tectoRNA. Two TLRs (a, blue; b, orange) bind to the TL (green) in two orientations. Helical segments are denoted by cylinders and loops/bulges by solid lines connecting them; tertiary contacts are depicted as dashed red lines. The binding of TLR_a (blue) aligns the orthogonal TL • TLR pair (black) for binding, whereas binding of TLR_b (orange) puts the tectoRNA in a conformation that does not favor the orthogonal TL • TLR. (B) Different scaffold geometries can induce tectoRNA conformation changes during binding. The TL • TLRs in (A) are placed into two example tectoRNA scaffolds, one with a straight helix (Scaffold 1) and one with a bulge (Scaffold N). When the geometries of the scaffold and TL • TLR align, tectoRNA interaction is stronger ( < ); when they do not align, the tectoRNA must change its conformation, resulting in unfavorable binding ( > ). (C) Comparison of thermodynamic fingerprints for two TLRs with different binding geometries. The ΔG values (top) across different scaffolds make up their respective thermodynamic fingerprints. ΔΔG_rel values (red) are used to compare the thermodynamic fingerprints of different TLR sequences. They are obtained from comparing the ΔΔG between the two sets of ΔG values (bottom) to the median ΔΔG value (gray). (D,E) Comparisons of the thermodynamic fingerprints of the 11ntR with the IC3R (red) and Vc2R (orange) binding to a (D) GAAA or (E) GUAA TL. Median and 95% CIs of ΔΔG_rel are depicted across the 20 scaffolds. Scaffolds that induce a significantly different binding conformation (ΔΔG_rel ≠ 0) are denoted by black arrows. (F) The values compared against the 11ntR (top) or IC3R (bottom) for all 11ntR → IC3R variants. Median and 95% CIs for are plotted. Red triangles represent variants with significant differences from the 11ntR; blue triangles represent variants with significant differences from the IC3R; white diamonds represent variants with significant differences from both. (G) The values compared against the 11ntR (top) or Vc2R (bottom) for all 11ntR → Vc2R variants. Median and 95% CIs for are plotted. Orange, blue, and white symbols represent variants with significant differences from the 11ntR, Vc2R, or both, respectively.