Template-dependent DNA ligation for the synthesis of modified oligonucleotides

Chemical modification of DNA is a common strategy to improve the properties of oligonucleotides, particularly in the context of therapeutics and nanotechnology. Existing synthetic methods essentially rely on phosphoramidite chemistry or the polymerization of nucleoside triphosphates and are limited in terms of size, scalability, and sustainability. Herein, we report a robust alternative method for the de novo synthesis of modified oligonucleotides using template-dependent DNA ligation of shortmer fragments. Our approach is based on the fast and scaled accessibility of chemically modified shortmer monophosphates as substrates for the T3 DNA ligase. This method has shown high tolerance to chemical modifications, flexibility and overall efficiency, thereby granting access to an ultimately broad range of modified oligonucleotides of different lengths (20 →160 nucleotides). We have applied this method to the synthesis of clinically relevant antisense drugs and highly modified ultramers. Furthermore, the designed chemoenzymatic approach has great potential in numerous applications in oligonucleotide therapeutics, bioorganic chemistry, pharmacology, and chemical biology.