Base-modified nucleotides as substrates for TdT-based enzymatic nucleic acid synthesis

A method of nucleic acid synthesis comprising:

• (a) providing a nucleic acid and a plurality of nucleoside triphosphates, wherein each of the plurality of nucleoside triphosphates comprises a modified base comprising a photocleavable carbon chain moiety having a length of at least 60 Å;
• (b1) contacting (i) the nucleic acid and (ii) a first nucleoside triphosphate of the plurality of nucleoside triphosphates with a first terminal deoxynucleotidyl transferase (TdT) to generate a first modified nucleic acid comprising the nucleic acid incorporated with one first nucleotide comprising the modified base from the first nucleoside triphosphate;
• (c1) photocleaving the photocleavable carbon chain moiety of the modified base of the first nucleotide in the first modified nucleic acid to remove the photocleavable carbon chain moiety from the first modified nucleic acid;
• (b2) contacting (i) the first modified nucleic acid with the photocleavable carbon chain moiety of the modified base of the first nucleotide removed and (ii) a second nucleoside triphosphate of the plurality of nucleoside triphosphates with a second TdT to generate a second modified nucleic acid comprising the first modified nucleic acid incorporated with one second nucleotide comprising the modified base from the second nucleoside triphosphate;
• and
• (c2) photocleaving the photocleavable carbon chain moiety of the modified base of the second nucleotide in the second modified nucleic acid to remove the photocleavable carbon chain moiety from the second modified nucleic acid.