Journal: Synthetic and Systems Biotechnology

Article Title: Semi-rational engineering of terminal deoxynucleotidyl transferase for high-efficiency enzymatic de novo DNA synthesis

doi: 10.1016/j.synbio.2026.03.009

Figure Lengend Snippet: Mutagenesis analysis of BtTdT variants. (a) L397 (green) or L397M (magenta) in BtTdT. (b) Activity test for M1. (c) R336/K338 in BtTdT (green) or R335/K337 in ZaTdT (orange). (d) Activity comparison of three BtTdT mutants. (e) Elongation by M3 with different iDNAs terminated with 16 dinucleotides and 4 types of 3′–ONH 2 –dNTPs. Reaction conditions: 1 mg/mL TdT, 1 μM iDNA, 42 °C, 10 min. “A∼, T∼, C∼, and G∼” stand for 3′–ONH 2 –dATP, 3′–ONH 2 –dTTP, 3′–ONH 2 –dCTP, and 3′–ONH 2 –dGTP, respectively. Note : Product bands at the same concentration may display different intensities due to sequence-specific effects.

Article Snippet: 3′–ONH 2 –dNTPs were bought from Firebird Biomolecular Sciences (Florida, USA), and 3′- O –CH 2 N 3 –dNTP and 3′–OCHCHCN–dNTP were purchased from Huana Biomedical Technology Co. Ltd. (Hefei, China).

Techniques: Mutagenesis, Activity Assay, Comparison, Concentration Assay, Sequencing

Journal: Synthetic and Systems Biotechnology

Article Title: Semi-rational engineering of terminal deoxynucleotidyl transferase for high-efficiency enzymatic de novo DNA synthesis

doi: 10.1016/j.synbio.2026.03.009

Figure Lengend Snippet: Further mutational work based on M3. (a) Key amino acids at the catalytic center in BtTdT (green) and ZaTdT (cyan). (b) Activity evaluation for M3-based mutants. (c) Extension of 3′–ONH 2 –dATP by Bt15AA R336L/K338G/L397M/E456G . Reaction conditions: 1 mg/mL TdT, 1 μM iDNA, 42 °C, 10 min. Note : Product bands at the same concentration may display different intensities due to sequence-specific effects.

Article Snippet: 3′–ONH 2 –dNTPs were bought from Firebird Biomolecular Sciences (Florida, USA), and 3′- O –CH 2 N 3 –dNTP and 3′–OCHCHCN–dNTP were purchased from Huana Biomedical Technology Co. Ltd. (Hefei, China).

Techniques: Activity Assay, Concentration Assay, Sequencing

Journal: Synthetic and Systems Biotechnology

Article Title: Semi-rational engineering of terminal deoxynucleotidyl transferase for high-efficiency enzymatic de novo DNA synthesis

doi: 10.1016/j.synbio.2026.03.009

Figure Lengend Snippet: Mutagenesis based on AF3-based structural analysis. (a) Position of surrogate substrate ATP in M3 variants with an amino acid substitution at E456. Enzyme-ligand complexes were predicted using AF3. E456S (yellow), E456G (aquamarine), E456 (green), and E456A (magenta). (b) Activity test of M4 mutant after adding 3′–ONH 2 –dATP for different time. (c) Position of surrogate substrate ATP in M4 variants with an amino acid substitution at D395. The distances in angstrom from the substrate's 3′-end to the reference residues E456 or D395 (wild-type) are shown in different color for each mutant. Enzyme-ligand complexes were predicted using AF3. D395 (M4, yellow), D395G (white), and D395M (red). (d) Extension of 3′–ONH 2 –dNTP by M4 in 5 min. Reaction conditions: 1 mg/mL TdT and 1 μM iDNA. (e) Low-energy binding conformations of 3′–ONH 2 –dNTP in the WT and three E456 variants, along with their structural overlays. The distances between the 3′-O atom in the WT conformation and those in the mutant conformations are indicated. (f) The average distance between the substrate's O–NH 2 moiety and the geometric center of the WT and three E456 mutant backbones. (g) Time course assays of M4 mutations at D395. (h) Extension of 3′–ONH 2 –dNTP by M5 in 30 s. Reaction conditions: 1 mg/mL BtTdT and 1 μM iDNA. (i) PAGE analysis of the 10 steps of extension by M5. Extension conditions: 1 mg/mL M5, 1 μM iDNA P2, 1 min. “A∼, T∼, C∼, and G∼” stand for 3′–ONH 2 –dATP, 3′–ONH 2 –dTTP, 3′–ONH 2 –dCTP, and 3′–ONH 2 –dGTP, respectively. In panels (e) and (f), blue, purple, pink, and orange denote wild type, E456A, E456G, and E456S, respectively. Note : Product bands at the same concentration may display different intensities due to sequence-specific effects.

Article Snippet: 3′–ONH 2 –dNTPs were bought from Firebird Biomolecular Sciences (Florida, USA), and 3′- O –CH 2 N 3 –dNTP and 3′–OCHCHCN–dNTP were purchased from Huana Biomedical Technology Co. Ltd. (Hefei, China).

Techniques: Mutagenesis, Activity Assay, Binding Assay, Concentration Assay, Sequencing