with the vacuum permeability µ0, the Planck constant h, the g-values of the electron and fluorine nucleus ge and gn, respectively, the Bohr magneton µB and the nuclear magneton µN. With these constants C amounts to C = 74.52 MHz Å 3 . For G5, a coupling constant Tread = 50 ± 7 kHz was read off that corresponds (Eq. 1) to a point-dipole distance of Rread = 11.5 ± 0.5 Å. The error was estimated from the width of the maxima of the peaks. For G43, a smaller splitting from a coupling constant Tread = 30 ± 4 kHz was observed, corresponding to a point-dipole distance of 13.5 ± 0.6 Å. For U46 no splitting could be observed, however a clear peak was still detected. To ensure that this signal of U46 did not arise from free F -interacting with the nitroxide label, a control experiment was performed. F ENDOR spectra of the free aptamer labelled in position G43 and U46 were measured in the presence of F -at W-band and Qband, respectively. In absence of Mg 2+ the F -binding site cannot form and indeed no ENDOR effect has been observed (Fig. ). Thus, all F ENDOR signals observed in the holo form arise from bound F -. To extract more information on conformational distributions from the ENDOR line shape we performed a spectral analysis in two steps. First, we simulated the spectra using our fast simulation routine SimSpec (see section 1 in SI) without any prior model, just assuming a Gaussian distribution of distances centred at Rread. The centre and the width of the Gaussian distribution were manually adjusted to minimize the residual between experiment and simulated spectrum (Fig. and). The simulations are displayed in Fig. (left). The Gaussian distance distribution reproduced the line shapes of G5 and G43 very well using a broadening (full width at half maximum, FWHM) corresponding to 2.4 Å. The simulated Tsim component resulted in Rsim values as given in Fig. (right) and in Table . For U46 a series of simulations was performed for Gaussian distributions of constant width 2.4 Å but varying Rsim between 18 and 28 Å. Examination of the residuals (Fig. and Fig. ) suggested that Rsim ≳ 20 Å can generally be considered a lower boundary for the distance, when no splitting occurs. We note that this is the first detection of a F -nitroxide distance ≳ 20 Å, demonstrating an extension of the accessible distance range. Indeed, the resolution of the splitting depended on the choice of the line width parameter. Our choice of a Lorentzian line of 7 kHz (slightly larger than the power broadening of the RF pulse) is based on an ongoing investigation of intrinsic ENDOR line widths. This resulted in Rsim ≳ 23 Å. Using a larger line width of 12 kHz, resulted in a shorter limit of Rsim ≳ 21 Å (Fig. ). Moreover, a single distance above 20 Å was not sufficient for reproducing the broad base of the ENDOR spectrum. This feature could be simulated by introducing an additional, shorter distance centred at about 16 Å and in a much smaller population (only 10% of the overall distance distribution). We note that the Mims ENDOR experiment enhances the contribution of shorter distances, making such a small contribution discernible in the spectrum. In order to examine to what extent conformational distributions affect the spectra, we also performed simulations of rigid Pake patterns based on a single Rsim value convoluted with the estimated intrinsic ENDOR line width. Comparison of spectral simulations (Fig. , Fig. ) showed indeed additional broadening caused by a distance distribution between F -and spin label, which was most pronounced for the shortest distance in G5. In the second step of analysis, we estimated the contribution of the spin label to the ENDOR line width by rotamer modelling of label orientations in MtsslSuite and MMM (Fig. ), based on the available crystal structure (PDB 4ENC, see section 1 in SI). This modelling produces rotamer ensembles of spin label orientations as represented in Fig. . For each rotamer, distance and orientation were considered. The distance was taken from the midpoint of the NO-bond to the F -while the orientation was defined through the Euler angles α and β between the dipolar tensor and the spin label g-tensor, as illustrated in Fig. . The Mims ENDOR spectrum for each of up to 400 rotamers was computed in SimSpec (see section 1 in SI) and then added to generate the sum ENDOR spectrum (Fig. ). Both diastereomers were considered, however the modelling does not predict their relative weight. For G5, both diastereomers were considered in equal weight, while for G43 only one diastereomer was considered, as the second cannot be populated due to steric clashes. For U46, the weight of the diastereomer leading to the shorter distance was adjusted to reproduce the ENDOR spectrum. Starting from the construct labelled in position G5 we obtained a substantial deviation of 3 Å between the predicted Rmodel and the observed Rsim distance. However, the distance distribution width with ΔR = 2.1 Å was very close to the simulated value of 2.4 Å (Fig. ). For construct G43 we found a good agreement between the Gaussian and the predicted distance distribution. For U46 a small deviation in the long distance (Rmodel,1) could be observed while the width of this distribution contribution was in good agreement with the Gaussian. For all three constructs, we found that the width of the distance distributions predicted by the rotamer modelling, when keeping a rigid body of the RNA, was consistent with the width extracted from the model-free Gaussian distributions. This means that no substantial broadening arising from RNA backbone heterogeneity is observed in the ENDOR spectra. This is a significant result, which differed from the PELDOR findings, where the experimental distance distributions were considerably broader than the ones predicted from rotamer modelling. This is important, as it is known that rigid biomolecules (e.g., model protein GB1) allow reconciling PELDOR and RIDME data and ENDOR data all based on a single protein structure. Furthermore, considering the mean distances, we observed good agreement only for G43 and U46. At G5, the significant deviation between the experimental and modelled distances can be rationalised by crystal contacts in the 5' region (Fig. ) not being present in solution (vide infra). We conclude that the ENDOR data, although sensitive to small distance shifts of a few angstroms, do not report structural heterogeneity of the holo aptamer in the vicinity of the binding pocket. This indicates that the RNA structure around the active site is well-defined in the holo form.