Processes for the production of oligonucleotides

A process for producing a single-stranded oligonucleotide product, wherein the single-stranded oligonucleotide product comprises at least one modified nucleotide residue selected from the group consisting of modification at the 2′ position of the sugar moiety, modification of the nucleobase, and modification of the backbone of the nucleotide residue, wherein the process comprises:

• a) providing a pool of oligonucleotides comprising segments of the single-stranded oligonucleotide product, wherein at least one segment comprises the at least one modified nucleotide residue, and wherein at least one segment is produced by enzymatic synthesis using a single-stranded ligase (ssLigase);
• b) providing a template oligonucleotide having a sequence complementary to the sequence of the single-stranded oligonucleotide product, wherein the template oligonucleotide has a property that allows it to be separated from the single-stranded oligonucleotide product;
• c) contacting the pool of oligonucleotides with the template oligonucleotide in conditions to allow annealing of the segments to the template oligonucleotide;
• d) joining the segments by enzymatic ligation with a ligase to form the single-stranded oligonucleotide product annealed to the template oligonucleotide and impurity oligonucleotide strands annealed to the template oligonucleotide;
• e) changing the conditions to denature the annealed template oligonucleotide and any impurity oligonucleotide strands, and separating the impurity oligonucleotide strands from the annealed template oligonucleotide and single-stranded oligonucleotide product;
• f) changing the conditions to denature the annealed template oligonucleotide and the single-stranded oligonucleotide product, and separating the single-stranded oligonucleotide product;
• and
• g) recycling the template oligonucleotide,
• wherein a process for producing the at least one segment using a ssLigase comprises:
• (i) adding a 3′,5′ nucleotide bisphosphate, having at least one phosphate oxygens substituted by sulphur, to the 3′-OH of a single-stranded oligonucleotide primer by using the ssLigase;
• (ii) removing the 3′-phosphate, 3′-thiophosphate or 3′-dithiophosphate by using a phosphatase;
• (iii) repeating steps (i) and (ii) to produce the segment attached to the single-stranded oligonucleotide primer;
• (iv) releasing the segment from the single-stranded oligonucleotide primer by using a sequence specific endonuclease.