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. 2020 Jan 16;11(1):102.
doi: 10.3390/genes11010102.

Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis

Affiliations

Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis

Sebastian Barthel et al. Genes (Basel). .

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.

Keywords: DNA data storage; TdT; enzymatic DNA synthesis; oligonucleotide synthesis; polymerase cofactors; secondary structures; template-independent polymerase; terminal deoxynucleotidyl transferase; thermostability engineering.

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Conflict of interest statement

S.P. and D.H.A. are co-founders of Ansa Biotechnologies, Inc., a company commercializing the polymerase-nucleotide conjugate technology. S.P., D.H.A., and S.B. are currently employed by Ansa Biotechnologies, and all authors have a financial interest in Ansa Biotechnologies. J.D.K. and N.J.H. have a financial interest in Ansa Biotechnologies.

Figures

Figure 1
Figure 1
(A) Elongation of DNA primers with 3′ terminal hairpins of varying length with ddTTP using MTdTwt in RBC reaction buffer (50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, 0.25 mM cobalt chloride, pH 7.9) at 37 °C (n = 3 replicates). Elongation rates were normalized to rates on the unstructured substrate (0 bp). (B) Elongation of an unstructured primer (P1) with ddNTPs using MTdTwt in reaction buffer (50 mM potassium acetate, 20 mM Tris acetate, pH 7.9) with varying M2+ concentrations: RBC (10 mM Mg2+, 0.25 mM Co2+), RBC without Mg2+ (0.25 mM Co2+), and TP8C (1 mM Co2+) (n = 2 replicates). Elongation rates were normalized to rates in RBC. (C) Elongation of an 8 bp hairpin primer (P5) with ddTTP or ddGTP in RBC and TP8C using MTdTwt (n = 3 replicates). Elongation rates were normalized to rates in RBC. In all of the above, error bars correspond to mean ± SD; T = ddTTP; A = ddATP; C = ddCTP; G = ddGTP.
Figure 2
Figure 2
Optimizing the divalent cation concentrations and elevating the reaction temperature by 10 °C synergistically increased the incorporation rates of free and conjugated nucleotides into a hairpin primer. Elongation of hairpin primer P5 by either free ddTTP and MTdT-evo or MTdTc302-evo-dTTP conjugates were performed in RBC and TP8C buffer at 37 °C and 47 °C. Elongation rates were normalized to rates in RBC at 37 °C. Error bars correspond to mean ± SD of n = 3 independent replicates.

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