Orthogonal Ligase Enzymes for Assembly of Modified Nucleic Acids

Modified nucleic acids including antisense oligonucleotides and siRNAs are set to transform medicine. Currently there are 18 approved drugs, and over 150 modified nucleic acid therapeutics currently in clinical trials for the treatment of cancer, cardiovascular, and neurodegenerative conditions as well as other diseases. The mRNA vaccines used to combat COVID-19 and related cancer vaccines also comprise of modified mRNA with alternative nucleobases. In addition, modified nucleic acids are widely used in basic research (structure-function studies). Currently, nucleic acids are mainly produced by outdated solid-phase synthesis (SPS), which requires many steps per cycle and is highly atom inefficient, using expensive monomers, deleterious reagents and large amounts of organic solvents. Such methods are very difficult to scale up and create a huge amount of waste which has hampered drug production. Longer modified mRNA vaccines can be produced using template-dependant polymerase enzymes, but this limits the extent to which modifications can be included. In OLIGNA I aim to overcome these limitations and deliver an alternative template independent enzymatic approach for the synthesis of modified nucleic acids which is more sustainable than SPS, in addition to being more versatile than existing enzymatic approaches. The project is comprised of three work packages: WP1 aims to engineer orthogonal ligases enzymes to insert modified nucleotide monomers between two RNA strands to generate longer site-specifically labelled RNAs for structure-functional studies; WP2 will explore developing the orthogonal ligases for the iterative assembly of native RNA; WP3 aims to further engineer the ligases to accept modified nucleotide and dinucleotide precursors including the common 2´-sugar modifications required for the production of nucleic acid therapeutics. OLIGNA will deliver game-changing technology to transform nucleic acid synthesis, providing new research tools and medicines.