Figure 6.

Oligonucleotide synthesis using DNA polymerases. (a) An approach exploiting an engineered 9°N polymerase and a universal template that contains all possible three base combinations with a 3′-deoxyuracil base. Following transient hybridization of the template to adjacent stands, the 3′-end is extended using 3′-O-azidomethyl-modified nucleotides. Following base addition, the reversible terminator is removed with TCEP to generate a free 3′-OH group which can undergo further extensions. The final oligonucleotide product is cleaved from the template at the deoxyuracil site using uracil-DNA glycosylase and apurinic/apyrimidic endonuclease. (b) A terminal deoxynucleotidyl transferase (TdT) approach for oligonucleotide synthesis. A R335L K337G variant of ZaTdT was used to extend an initiating sequence immobilized on steptavidin using 3′-ONH₂-modified nucleotides. Following the extension step, the 3′-ONH₂ blocking group is cleaved using sodium nitrite buffer.