An enzyme cascade enables production of therapeutic oligonucleotides in a single operation

Abstract

Therapeutic oligonucleotides have emerged as a powerful drug modality with the potential to treat a wide range of diseases; however, the rising number of therapies poses a manufacturing challenge. Existing synthetic methods use stepwise extension of sequences immobilized on solid supports and are limited by their scalability and sustainability. We report a biocatalytic approach to efficiently produce oligonucleotides in a single operation where polymerases and endonucleases work in synergy to amplify complementary sequences embedded within catalytic self-priming templates. This approach uses unprotected building blocks and aqueous conditions. We demonstrate the versatility of this methodology through the synthesis of clinically relevant oligonucleotide sequences containing diverse modifications.

Recent advances in nanoparticulate RNA delivery systems

“…siRNA modification is a mature technology that has led to Food and Drug Administration (FDA)-approved products including Onpattro, an siRNA lipid nanoparticle (LNP) drug, and multiple naked ligand-conjugated siRNAs ( 1 ). While chemical synthesis of small RNAs has been the primary strategy, enzymatic synthesis of custom-modified siRNA has recently been reported which could possibly be more suitable for manufacturing siRNA at scale ( 2 ). Chemical modifications of gRNAs for CRISPR gene editing can enhance editing efficiency ( 3 – 7 ).…”

An Update on Protection of 5′‐Hydroxyl Functions of Nucleosides and Oligonucleotides

“…In this competition, chemical synthesis retains its value and in fact it takes a strong lead, particularly when the demand is for large numbers or large quantities of oligonucleotides. Recently, an exciting biocatalytic strategy has been described for the assembly of therapeutic oligonucleotides offering an alternative option for improved sustainability through reactions performed in water (Moody et al, 2023).…”

Template-dependent DNA ligation for the synthesis of modified oligonucleotides

“…1C). [23][24][25][26][27][28][29][30][31][32] However, most current methods are restricted either in terms of sequence length, possibility of site-specific introduction of modified nucleotides, speed of production, and/or sustainability.…”

“…It is also worth mentioning that the synthesis of antisense drugs by ligation strategy could be potentially compatible with the use of self-priming templates and restriction endonuclease as demonstrated by the Lovelock group for polymerases. 24…”