Assembly of nucleic acids with site-specific modifications

The ability to synthesise large nucleic acids with site-specific chemical modifications would be invaluable for applications including therapeutics, epigenetics, and nanotechnology. Current approaches mainly rely on polymerase enzymes or solid-phase chemical synthesis, both of which have their limitations. The former is restricted to enzyme-tolerated modifications and cannot achieve site-specificity beyond the level of Watson-Crick base pairing; the latter is constrained to oligonucleotide lengths of around 150 nucleotides for DNA (75 nucleotides for RNA) due to imperfect monomer coupling. To address these issues, ligation of chemically synthesised oligonucleotides is investigated for the assembly of long nucleic acids (beyond 100 nucleotides) with site-specific modifications. The limits of enzymatic ligation are determined by optimising DNA and RNA assembly using ligases, before chemical ligation is explored as a potentially more scalable and cost-effective alternative. In particular, copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) is investigated for the chemical ligation of RNA oligonucleotides. The reaction produces an unnatural triazole backbone at the ligation site. While the triazole backbone has been shown to be tolerated by DNA and RNA polymerases in replication and transcription, it has been unexplored for translation. An assay that involves rolling circle translation of a circular mRNA template, assembled by CuAAC and enzymatic ligation, is developed using a prokaryotic cell-free protein synthesis system. The effects of the local sequence environment and triazole position relative to the codon reading frame are examined, with special attention placed on the codon wobble position. Prokaryotic ribosomes are able to read triazole-containing mRNA to produce polypeptide translation products. The yields are lower than that of the natural backbone control, possibly due to destabilised hybridisation between the tRNA and mRNA, a feature that requires further optimisation.