UC Berkeley researchers describe a new enzymatic oligo synthesis method in Nature Biotechnology
Most oligonucleotides used in research and therapeutics are synthesized using the phosphoramidite method. However, since this method limits the length of DNA synthesized to ~ 200 nt/oligo and also generates hazardous waste, people have been trying for years to develop an enzymatic DNA synthesis method. In a recent issue of Nature Biotechnology, Palluk et al. from the Keasling lab at UC Berkeley performed a proof-of-concept study for a new method involving reversible tethering of a nucleotide to the polymerase terminal deoxynucleotidyl transferase (TdT).
TdT is a polymerase that can add dNTPs to the 3’ end of an oligo in a sequence-independent manner. The trick here was to use it to incorporate just one dNTP per round of synthesis by physically tethering the enzyme to the nucleotide, blocking addition of another nucleotide. The first TdT-nucleotide conjugate they made used a PEG linker and a disulfide bond that could be broken with 2-mercaptoethanol, and was able to extend a DNA primer by ~ 1 nucleotide. For the next version they used a propargylamino dNTP and a linker with a photocleavable bond, which leaves a smaller scar on the incorporated nucleotide after cleavage. They demonstrated the enzyme-DNA complex formation by staining a gel with One-Step Lumitein™ UV for the protein and FAM for the DNA; the overlapping signal reverted to separate bands after photo-cleavage of the complex.
Using this type of conjugate, they demonstrated that they could perform 10 cycles of addition and cleavage to achieve the oligonucleotide sequence of interest, and that DNA synthesized in this manner was amenable to downstream applications such as PCR and NGS. However, further optimization will need to be done to improve the error rate and reaction time so that this method can be employed in the synthesis of long oligos.
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