Extended Data Fig. 1: Properties of used constructs and validation of applied modifications.

a) Table of used constructs. b) Gel electrophoretic analysis of substrate cleavage of indicated Dz variants (top, using fluorescein-labelled RNA) and of effects of 2ʹF stabilization (bottom, using GelRed staining). c) Schematic picture of 2ʹF RNA modification (left) and affected protons in [¹H,¹H]-TOCSY fingerprint spectra (Dz^5C–RNA^2ʹF, blue and Dz^5C–RNA, black). The 2ʹF modification at rG₀ induces CSP only in its direct proximity, i.e., at the H5 and H6 position of rU_-1. While the absence of ¹³C enrichments in the RNA substrate impedes accurate determination of potential effects of the 2ʹF modification on the pseudorotation phase around the cleavage site⁴⁸, analysis of the CSP pattern induced by the 2ʹF modification at all resolved ¹H positions within the complex (d) confirms that the substrate stabilization does not alter the overall structure of the precatalytic complex. e) Nucleotide-specific ratio of peak intensities in the presence and absence of 1 mM Mg²⁺ for Dz^5A–RNA^2ʹF (red) and Dz^5C–RNA^2ʹF (blue). To enable a reliable comparison between different nucleotides, the changes of the cross-peak intensities of the correlation between H1ʹ and H6/H8 are shown for each Dz nucleotide. The peak disappearing in the loop region is linked to exchange processes occurring in the NMR intermediate-exchange regime, whereas the otherwise observed CSPs reveal exchange processes in the NMR fast-exchange regime. It can be concluded that Mg²⁺ resides longer within the catalytic loop of the 5A variant, possibly facilitating cleavage. f) Validation of labelling efficiency and cleavage capabilities of the click chemistry approach used for PRE spin labelling. A schematic of the used copper-catalysed click reaction using TEMPO-azide (2) and 5-ethynyl-2’-deoxyuridine (EdU) (1) (f, top). The EdU was used to replace one selected thymine nucleotide in the Dz sequence. To test labelling efficiency and cleavage capabilities, a FAM-azide was used, enabling direct detection via SDS-PAGE (f, bottom). To evaluate the labelling efficiency, identical amounts of a commercial FAM-labelled Dz were loaded on the indicated lane. g) Validation of activity of Dz^6xF. Denaturing SDS-PAGE results of time-dependent substrate cleavage of Dz^5C without and with the six 2ʹ-¹⁹F substitutions (Dz^6xF). The data demonstrate that the fluorine atoms do neither affect the Dz’s cleavage activity nor Mg²⁺-dependency.