The fluoride binding riboswitch provides a unique opportunity to measure inter-spin distances to the active site in the holo aptamer, containing an endogenous F -, by F ENDOR spectroscopy. For this, F Mims ENDOR (pulse sequence in Scheme 2b) was performed with three constructs containing a single nitroxide label at positions G5, G43, or U46 (Fig. ). F ENDOR spectra of G5 and G43 were measured at W-band (94 GHz) and four different excitation positions in the EPR spectrum (Fig. , left). The orientationally selected ENDOR spectra were weighted by the spectral intensity at the excitation position, added, and analysed (see section 1 in SI). For construct U46, spectra were measured at Q-band (34 GHz), taking advantage of superior concentration sensitivity, as the expected distance was longer (Fig. ). At Q-band, H ENDOR signals from the spin label methyl groups overlap with the F ENDOR signals (Fig. ). This was circumvented by introducing a deuterated nitroxide spin label (see section 1 in SI). Furthermore, this construct showed the slowest echo decay (i.e., slowest transverse dephasing, Fig. ) contributing to improved sensitivity. At 34 GHz 19 F ENDOR spectra were measured only at two different spectral positions of the EPR line (Fig. , right), and no significant effect of orientation selection could be observed within the achievable resolution and signal-to-noise ratio. The three F ENDOR spectra of the holo riboswitch are displayed in Fig. . For constructs G5 and G43, a well-resolved hyperfine splitting arising from the maxima of a Pake pattern was observed. From this splitting, called Tread, the perpendicular component T⊥ of the hyperfine tensor was estimated, giving first insight into the magnitude of the dipolar coupling and the corresponding inter-spin distance. Using the point-dipolar approximation the point-dipole distance (Rread) can be estimated as: 𝑇 𝑟𝑒𝑎𝑑 ≈ 𝑇 ⊥ = 𝜇 0 4𝜋ℎ ( 𝑔 𝑒 𝑔 𝑛 𝜇 𝐵 𝜇 𝑁 𝑅 𝑟𝑒𝑎𝑑 3 ) = 𝐶 𝑅 Eq. 1