Extended Data Fig. 1: Screening of oligonucleotide crosslinking chemistry.
From: Branched chemically modified poly(A) tails enhance the translation capacity of mRNA
(a) Summary of oligonucleotide chemical conjugation methods. Screening was performed using 15-nt poly-deoxyadenosine model substrates at micromolar concentrations. Modification handles were incorporated through oligonucleotide solid phase synthesis, followed by amine-NHS labeling and HPLC purification. (b) Gel electrophoresis of crude thiol-ene/yne oligonucleotide conjugation of 15-nt model substrates containing only one conjugation handle. (c) Gel electrophoresis of crude CuAAC and IEDDA 30-nt oligonucleotides bearing three alkyne/trans-cyclooctene handles reacting with 30-nt azide/tetrazine modified oligo. (d) Preliminary dual luciferase assay using branched mRNA prepared from CuAAC product mixture without HPLC purification using different equivalents of branched oligos versus stem oligo. Branching oligos contained modifications on the last six bases with either natural (5′ to 3′) or reversed (3′ to 5′) directionality. Time-course assay was performed as outlined in Fig. 1. n = 3 independent transfections for each construct. Mean ± s.e.m. P values were calculated by ordinary two-sided one-way ANOVA (comparison of means across time points). Gels are representative of at least two experiments.
