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

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.