Fig. 2. Reaction product characterization.

a. Anion exchange HPLC analysis of MTR1-catalysed reaction of 13-mer RNA substrate S (5'-AUACUGAGCCUUC-3') with m⁶G at 25°C for 23 h; 10 μM RNA (S), 12 μM MTR1, 100 μM m⁶G, 40 mM MgCl₂, pH 7.5. The ribozyme is labeled with Rz, the substrate RNA S and the reaction product P. HPLC traces of reference oligonucleotide S (= ref A), and the corresponding m⁶A and m¹A-modified synthetic RNAs are shown for comparison. m¹A-RNA elutes earlier, while A and m⁶A-RNAs cannot be distinguished. b. HR-ESI-MS of S* (top) and P* (bottom). Shown are the measured m/z spectra, the deconvoluted mass spectrum in red, and the simulated isotope pattern in grey (* denotes 3'-aminohexyl RNA) c. RNase T1 digestion and alkaline hydrolysis of reaction product P in comparison to S, m⁶A and m¹A references demonstrate that P contains m¹A. d. Atomic mutagenesis of RNA substrate. Individual data points (n = 6 for A, n = 2 for all others) and average (grey bar) shown. white: 17 nt RNAs, blue: 13 nt RNAs. Gel images and detailed description in Extended Data Fig. 4. e. Incubation under Dimroth rearrangement conditions (pH 10, 65°C, 1 h) produced m⁶A from m¹A, as seen in the + lanes of m¹A reference and MTR1 reaction product P.