Extended Data Figure 10. Analysis of XNA (FANA) substrates and enzymes prepared by solid-phase synthesis.

MALDI-ToF mass spectra showing expected masses of a, XNA (FANA) ligase substrate LigS1^F-3′phosphorylimidazolide (prepared by solid-phase synthesis of the 3′ phosphorylated (3′p) oligonucleotide, followed by reaction with carbodiimide and imidazole (see Methods)), b, XNA (FANA) ligase substrate LigS2^F, and XNAzymes c, FR17_6min, d, F2R17_6min, and e, FpImR4_2. f, Dephosphorylation assay of of versions of LigS1^F (3′ hydroxyl, lanes 1 and 2, 3′ phosphate, lanes 2 and 3, or 3′ phorphorylimidazolide, lanes 5 and 6) with calf intestinal phosphatase (lanes 2, 4 and 6). The majority of the LigS1^F preparation shown (~70%) is protected from dephosphorylation, consistent with formation of the 3′pIm. g, Urea-PAGE analyses of purified FANA substrates and XNAzymes.