Extended Data Fig. 2: Synthesis and characterization of topologically augmented branched mRNA.
From: Branched chemically modified poly(A) tails enhance the translation capacity of mRNA
(a-c) Representative HPLC purification and gel electrophoresis characterizations of branched oligonucleotides (oligo) containing 1-3 branching poly(A) tails. Fractions containing the desired products (boxed) were pooled and isolated. (d) Schematics of preparation of firefly luciferase (FLuc) mRNA constructs lacking poly(A) tail before ligation. FLuc mRNA without template encoded poly(A) tail was ligated to a scramble (non-polyA) stem oligo with 3′ end modifications (six phosphorothioate/2MOE at 3′ and terminal dideoxycytidine modifications) and internal alkyne (5-octadiynyl deoxyuridine or OU), with or without conjugation to 5′ azide labeled scramble or poly(A) branches with 3′ end modifications (the same as the stem oligo). (e) RNase H characterization of branched mRNA-oligo conjugates with branched poly(A) or scramble tails. The branching topology was confirmed by further band shift on TBU gel. (f) Branched poly(A) sequence, rather than chemical modifications alone, conferred enhanced protein expression over time. Relative FLuc luminescence was normalized to the scramble OU only oligo ligated mRNA at indicated time points. n = 3 independent transfections for each construct. Mean ± s.e.m. P values were calculated by two-sided unpaired t-test (with Welch’s correction) against the scramble OU + scramble azide construct at corresponding time points. (g) RNase H characterization of branched mRNA-oligo conjugates with multiple ploy(A) tails. mRNA with full-length hemoglobin UTRs and template encoded 100A-tail was ligated to 0 (mock ligation), 30 A, 60 A, or oligos with one, two, or three branched poly(A) tails and characterized by RNase H assay. The branching topology was confirmed by further band shift on TBU gel. Gels are representative of at least two experiments.
