From liquid-phase synthesis to chemical ligation: preparation of oligonucleotides and their backbone analogs in solution

Abstract

<p>The increasing need for sustainably produced oligonucleotides and their chemically modified counterparts has inspired numerous alternative scalable and greener production methods. Current manufacturing techniques depend on automated solid-phase synthesis and polymerase-based assembly, but liquid-phase synthesis and alternative engineered biocatalytic methods are emerging to meet the scale-up challenges. The scale, length, and modifications of the target nucleic acid product dictate the choice of protocol, which can be a combination of chemical and/or biocatalytic methods. While modern biocatalytic methods are competitive for the preparation of long oligonucleotides (up to 1 kb), liquid-phase synthesis is gaining a strong foothold for the large-scale preparation of short sequences (<30-mers), especially for the chemically modified therapeutic oligonucleotides. DNA-templated chemical ligation is an emerging technology expected to expand the range of liquid-phase synthesis to the production of longer sequences. This review focuses on oligonucleotide synthesis carried out exclusively in solution. Current protocols for liquid-phase synthesis of oligonucleotides and their backbone analogs, and template-assisted chemical ligation, yielding the phosphodiester linkage and its artificial mimics of biological relevance, are summarized.<br></p>