PELDOR distance measurements using the 4-pulse DEER sequence (Scheme 2 (a)) are particularly well suited to study structural or conformational changes. To map overall structure and preorganisation, PELDOR time traces were measured for the free, apo and holo aptamers for each of the four doubly spin labelled constructs. Sample preparation and experimental conditions for PELDOR are described in SI section 1. All spectra were recorded in frozen solution at 50 K. The time traces (Fig. ) displayed differently pronounced dipolar modulations, which indicated different widths of the respective distance distributions. The time traces were analysed using Tikhonov regularisation implemented in the software DeerAnalysis (Fig. ). All obtained distance distributions, despite being broad, showed a distinct most probable distance, but did not show significant differences between the free, apo and holo aptamers for the C14/C44, G29/G36, and A9/A49 constructs. The C4/C17 construct displayed the most pronounced dipolar modulation resulting in the narrowest distance distribution with the lowest uncertainty. Here, the free and apo forms provided no difference within uncertainty but the holo form yielded a slightly reduced most probable distance. These results could be confirmed by an alternative processing with a different implementation of Tikhonov regularisation in the software DeerLab and by neuronal network analysis with the software DeerNet (Fig. ). Interestingly, all distance distributions displayed significant distance probability up to 50 Å and beyond in all three processing approaches. Comparing the experimental distance distributions with the modelling performed to select these constructs revealed substantially broader distributions in the former. Nevertheless, all modelled distance probability is fully covered by the experimental distributions. Particularly, the DeerLab analysis hinted at the presence of multiple distances corresponding to multiple conformations. This illustrated a conformational flexibility of the RNA backbone that was not reproduced by rotamer modelling on the single conformer in the crystal structure. Interestingly, for all cases but C4/C17 the most probable distances (i.e., maxima of the experimental distributions) were shorter than the maximum of the modelled distributions. This indicated the presence of more compact structural arrangements in solution than in the crystal structure. The similarity in the PELDOR data between the free, apo, and holo forms confirmed the preorganisation of stems 1 and 2 in the free RNA and their retention in the apo and holo aptamers. The shortening of the C4/C17 distance in the holo compared to the free and apo forms was consistent with an increased formation of the pseudoknot only upon addition of F -in contrast to the findings for the B. cereus fluoride riboswitch. The long distances tailing up to 50 Å and beyond hint to an equilibrium involving a disordered or unfolded form of the aptamer. This was consistent with earlier fluoride binding ITC data that had been fitted to 0.75 and 0.87 binding sites suggesting 13-25% of aptamers not binding F -and contributing very broad peaks to the distance distributions of all three forms. ) spin labelled in silico with MtsslSuite and with arrows indicating the spin-spin distance assed by PELDOR measurements.