Scheme 1. RNA labelling at the phosphorothioate modification of the backbone using either a protonated or a per-deuterated spin label. The sugar 5' to the labelling site is modified to a 2'-deoxyribose to avoid strand scission. For F ENDOR, three constructs with one spin label each were chosen to result in linearly independent distance vectors between the respective label and the F -, allowing the trilateration of the F - position within the RNA structure (Fig. ). G5 and G43 gave respective calculated mean distances (Rmodel) of 8.5 Å and 13.5 Å to the F -, well resolvable below the 20 Å threshold. For U46 however, in silico labelling displayed a bimodal distance distribution. The longer Rmodel,1 was predicted to be at about 21.5 Å, likely too long to be resolved by F ENDOR. Nevertheless, the second, shorter Rmodel,2 of 15.5 Å was within the resolvable distance limit for F ENDOR with nitroxide labels. To ensure reproducible folding of the riboswitch and infer any perturbation of folding by the spin label, we established a folding protocol (see SI section 1) using 1 H NMR spectroscopy. This allowed to identify the free aptamer in absence of Mg 2+ and F -, the Mg 2+ -bound apo riboswitch, and the F --bound holo riboswitch in analogy to results for the B. cereus fluoride riboswitch. Comparison of 1 H NMR spectra in the imino regions of the free and holo aptamer for both, unlabelled and labelled RNA showed indistinguishable signals indicating appropriate folding after labelling (Fig. ). Alternatively, isothermal titration calorimetry (ITC) could be used to investigate labelled and mutated riboswitch constructs for retained aptamer functionality.