Extended Data Fig. 4: NMR-based structure determination of Dz^5C–RNA^2ʹF.

a,b) Experimental data and the resulting analysis of the PRE experiments. a) [¹H,¹H]-NOESY spectral extracts of a sample with a single TEMPO label at position T8 before (red) and after reducing the nitroxide spin label via the addition of ascorbic acid (black). b) The resulting intensity ratios between the two spectra for each resolved proton in the three samples. The marker size (in I/I₀ direction) indicates the error margin (SD) obtained from the analysis of values for all resolved cross peaks of the respective proton. Only protons with at least two different resolved cross peaks were considered. c) Subset of NOE-buildup curves used for the eNOE approach. Normalized NOE intensities recorded with mixing times between 40 and 800 ms (filled circles) and buildup curves (lines) determined by eNORA³². [¹H,¹H]-NOESY cross peaks for the same spin system occurring above (red) or below (green) the diagonal were considered for most NOE contacts. Notably, even short and geometrically fixed distances show considerable differences in their buildup behaviour, demonstrating the necessity to incorporate cross-relaxation effects. Calculated cross-relaxation rates (σ) and extracted eNOE distance range (within 20% error margin) (blue) are indicated. d–i)¹⁹ F-based NMR experiments. d) 1D ¹⁹F-NMR spectra of Dz^5C–RNA^2ʹF (top) and of Dz^5C variant containing six 2ʹ-¹⁹F modifications at position G₋₆, G₋₅, G2, C7, A11, and G14 (Dz^6xF, bottom) in complex with RNA^2ʹF. While [¹⁹F,¹⁹F]-NOESY spectra of Dz^6xF–RNA^2ʹF did not show any detectable peaks (data not shown), ¹H-detected (e) or ¹⁹F-detected (f) [¹H,¹⁹F]-HOESY spectra show a limited number of distinct cross correlations that were used for sequential resonance assignments as well as long-range distance restraints. g) H1ʹ and H2ʹ protons of the respective fluorinated ribose moieties can be identified in a [¹H,¹H]-NOESY spectrum via the peak splitting induced by the strong J_FH coupling. h+i) To overcome sensitivity limitations of the 2D HOESY correlations, ¹⁹F-saturated and ¹H-detected STD NMR was used. h) Resulting ¹⁹F-STD spectrum of Dz^5C–RNA^2ʹF. i) ¹⁹F-STD spectra of Dz^6xF–RNA^2ʹF using the indicated ¹⁹F saturation frequencies (color code refers to assignment in panel d). j-m) Characterization of residual dipolar couplings (RDCs). j) Example of RDC-induced frequency shifts in the precatalytic complex. The section shows the H5-C5 cross peak of loop position C13 in a [¹H,¹³C]-HSQC spectra recorded in the absence (black) and presence (red) of Pf1 phage at 20 °C using ¹³C isotope-labelled Dz^5C in complex with unlabelled RNA^2ʹF. k) 1D cross section of the cross peaks shown in j) representing experimental limitations due to linewidths, peak overlap and signal-to-noise effects. l,m) Correlation plot of observed and back-calculated RDC constants for a non-matching structure of cluster III (l) and the improved correlation of the structure in cluster I (m). Although experimental limitations introduce larger error margins in the determined values (as visible by RMSD values), an RDC effect is still apparent (as visible by a considerably increased correlation).