Fig. 2. Representative examples of splint-mediated enzymatic and non-templated chemical cyclisation reactions to give the cyclic oligonucleotide templates 1_PO4 (A), 1_Tz (B), 1_PA (C) and 1_Am (D). The crude reaction mixtures were analysed by polyacrylamide gel electrophoresis on a 15% denaturing gel. Samples were imaged by UV shadowing. (A) Enzymatic cyclisation of linear unmodified 5′-phosphate/3′-hydroxyl oligonucleotide using T4 DNA ligase. (B) CuAAC-mediated cyclisation of linear 5′-azide/3′-alkyne oligonucleotide, introducing a triazole linkage. (C) Di-imide-mediated cyclisation of linear 5′-amine/3′-phosphate oligonucleotide, introducing a phosphoramidate linkage. (D) Di-imide-mediated cyclisation of linear 5′-amine/3′-carboxyl oligonucleotide, introducing an amide linkage. (i) = linear oligonucleotide substrate; (ii) = crude cyclisation reaction. *The major, slow-migrating side-product observed in the crude reaction mixture for the enzymatic ligation reaction is assigned as a cyclic dimer (molecular weight determined by UPLC-MS: 30 915 Da; calculated molecular weight for cyclic dimer: 30 912 Da; see Fig. S10, ESI†).
