Abstract
Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3′ terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin.
Highlights
Oligonucleotide synthesis is essential for modern biological research and promises to enable new “digital biology” applications, such as DNA-based data storage and computation [1]
Elongation rates were to rates on the unstructured substrate (0 bp). (B) Elongation of an unstructured primer (P1) with normalized to rates on the unstructured substrate (0 bp). (B) Elongation of an unstructured primer ddNTPs using MTdTwt in reaction buffer (50 mM potassium acetate, 20 mM Tris acetate, pH 7.9)
We demonstrated that wild-type terminal deoxynucleotidyl transferase (TdT) substantially reduced activity on primers with 30 terminal structure and we described two synergistic approaches for improving its activity on these substrates: Optimizing the divalent ion concentrations and elevating the reaction temperature
Summary
Oligonucleotide synthesis is essential for modern biological research and promises to enable new “digital biology” applications, such as DNA-based data storage and computation [1]. There is only one method available for oligonucleotide synthesis: The nucleoside phosphoramidite method [2]. This method has several key disadvantages for emerging DNA applications, including that (1) it is limited to the direct synthesis of high-quality ~250 mers [2] and (2) it strictly uses anhydrous solvents and produces a toxic and flammable waste stream, making it burdensome to operate in new settings, such as a data center or a spacecraft [3].
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