1. Overview

Throughout the engineering design cycles of our project, team LIUAN-Nanjing designed and characterized 9 new basic parts and 8 new composite parts. We also provided a reversible 3’-O-(2-nitrobenzyl)-modified nucleotides to enable the ordered and controlled enzymatic synthesis of DNA.

Table 1: Parts Overview

Part Number	Type	Description	Length
BBa_K5392000	Basic	ZaTdT-wild type	1548bp
BBa_K5392001	Basic	ZaTdT-mutant type (ZaTdT-R335P)	1548bp
BBa_K5392002	Basic	ZaTdT-mutant type (ZaTdT-R335W)	1548bp
BBa_K5392003	Basic	ZaTdT-mutant type (ZaTdT-R335I)	1548bp
BBa_K5392004	Basic	ZaTdT-mutant type (ZaTdT-R335F)	1548bp
BBa_K5392005	Basic	ZaTdT-mutant type (ZaTdT-R335M)	1548bp
BBa_K5392006	Basic	ZaTdT-mutant type (ZaTdT-K337A)	1548bp
BBa_K5392007	Basic	ZaTdT-mutant type (ZaTdT-K337L)	1548bp
BBa_K5392015	Composite	Vector-ZaTdT-KanR	6832bp
BBa_K5392016	Composite	Vector-ZaTdT-R335P-KanR	6832bp
BBa_K5392017	Composite	Vector-ZaTdT-R335W-KanR	6832bp
BBa_K5392018	Composite	Vector-ZaTdT-R335I-KanR	6832bp
BBa_K5392019	Composite	Vector-ZaTdT-R335F-KanR	6832bp
BBa_K5392020	Composite	Vector-ZaTdT-R335M-KanR	6832bp
BBa_K5392021	Composite	Vector-ZaTdT-K337A-KanR	6832bp
BBa_K5392022	Composite	Vector-ZaTdT-K337L-KanR	6832bp

2. Engineering Cycle 1

2.1. Stage 1: Design—Selection Of TdT Orthologs And Nucleotides Modification

As a promising technology, enzymatic DNA synthesis has been gaining interest since the 1950s. Terminal deoxynucleotidyl transferase (TdT), one of the most promising DNA polymerases for de novo DNA synthesis, has been known to be able to incorporate random nucleotides into initiator strands in the absence of a template since the 1960s[1-2]. However, the interest in developing enzymatic DNA synthesis declined after the successful establishment of the phosphoramidite method during the 1980s. In recent years, TdT has again attracted considerable attention due to the need for faster synthesis of longer DNA strands for data storage and synthetic biology[3-5].

The major challenge in the application of TdT for the programmed synthesis of artificial DNA lies in controlling the polymerization of user-defined nucleotides. Currently, reversible nucleotide terminators have been modified to control DNA synthesis[6]. Among the functional modifications, reversible chemical moieties capping the 3’-OH group of the sugar unit have garnered great interest due to their better termination effect[7]. However, TdT has a relatively small active cavity, making it more difficult to incorporate sugar modified nucleotides than natural nucleotides[8-9]. The successful incorporation of sugar-modified nucleotides requires extensive engineering of TdT[10]. Therefore, the implementation of a 3’-O-blocked reversible terminator and TdT for de novo DNA synthesis relies on the selection of a suitable blocking group, engineering of TdT, and optimization of cycling conditions.

The strategy of employing TdT for DNA synthesis relies on the stepwise addition of desired nucleotides. This can be accomplished by adopting a reversible terminator, which stop the synthesis following the addition of each new nucleotide analogue. A wide range of sugar-modified nucleotide terminators have been successfully applied to pause nucleotide incorporation in DNA sequencing[11]. Here, we designed a 3’-O-(2-nitrobenzyl)-dNTP that is a 3’-O-blocked reversible terminator.

The current knowledge regarding the structural features and physiologicalfunctions of template-independent DNA polymerases is primarily derived nucleotide addition by TdTs derived from different species has remained elusive[12]. A recent study based on the phylogenetic tree of 137 TdT genes across the vertebrate taxa, selected 14 TdTs from major clades of vertebrates including fish, amphibians, mammals, reptiles, and birds to test their catalytic activities in the incorporation of nucleotides into single-stranded initiators.They found that ZaTdT from Zonotrichia albicollisnhad the highest polymerization activity for natural nucleotide among the tested TdTs. Therefore, we chose ZaTdT for the subsequent experiments.

2.2. Stage 2: Build—Plasmids Construction

We incorporated the sequences of the target gene into the pET28a vector. Then the vector plasmid was transfected into E.coli DH5-alpha competent cells for purification and amplification. In this way, plasmids with the ZaTdT gene were obtained.

2.3. Stage 3: Test—catalytic activity assay of ZaTdT.

We transfected the ZaTdT gene-bonded pET28a plasmid into E.coli BL21(DE3) competent cell. After overnight, Colonies were then picked and performs protein expression. We identified the ZaTdT expression by SDS-PAGE and tested the incorporation of modified nucleotides by purified ZaTdT with polyacrylamide gel electrophoresis.

2.4. Stage 4: Learn

Through this cycle, we learned how to use SnapGene to construct plasmid and the entire process of protein expression and purification as well as catalytic activity testing. Polyacrylamide gel electrophoresis (PAGE) can be used to observe initiator strand extension. Our experiments showed that ZaTdT displayed the highest incorporation efficiency to 3’-O-(2-nitrobenzyl)-dTTP and Slightly activity to 3’-O-(2-nitrobenzyl)-dCTP. Furthermore, other 3’-O-blocked reversible terminator showed no observable extension when used wild type ZaTdT.

3. Engineering Cycle 2

3.1. Stage 1: Design—Protein Engineering of ZaTdT

To make the active center more compatible with 3’-O-(2-nitrobenzyl)-modified nucleotides, we attempted to reshape the catalytic cavity using mutagenesis on the residues within 6 Å around 3’-O-(2-nitrobenzyl) and the base moiety. We predicted the 3D structure of ZaTdT by homology modelling and molecular docking it with 3’-O-(2-nitrobenzyl)-dATP. The results of molecular docking indicate that residues Arg335 and Lys337 are closely related to the catalytic activity of ZaTdT.

Analysis of the molecular dynamics (MD) trajectories showed that R335 and its spatially neighboring residue K337 in ZaTdT contacted the triphosphate group through hydrogen bonds. We anticipate that eliminating the hydrogen bonds binding force between the above residues and the triphosphate group will release the greater freedom of the modified nucleoside within the active pocket, thereby increasing catalytic activity. Consequently, We performed molecular dynamics simulations of the 3’-O-(2-nitrobenzyl)-dATP docked into ZaTdT, ZaTdT-R335P, ZaTdT-R335W, ZaTdT-R335I, ZaTdT-R335F, ZaTdT-R335M, ZaTdT-K337A and K337L. The predicted results show that above substitution can abolish the hydrogen-bond connection at position 335 or reduce the frequency of hydrogen-bond formation between the triphosphate group and Lys337.

3.2. Stage 2: Build—Plasmids Construction

To obtain the desired saturation mutagenesis, oligonucleotide primers were designed with degenerate codons. In addition, each single-site saturation mutant was generated according to the PCR-based QuickChange method. The PCR was performed according to the operation manual. The PCR product was digested with DpnI restriction enzyme and transformed into E.coli DH5-alpha competent cells.

3.3. Stage 3: Test—catalytic activity assay of ZaTdT Mutant.

We transfected the Sequencing is correct ZaTdT mutant plasmid into E.coli BL21(DE3) competent cell. After overnight, an appropriate colony was used to express the mutant protein and verify its activity.

3.4. Stage 4: Learn

Through this cycle, we learned how to use homology modelling to predict the 3D structure of protein and use molecular docking to excavate closely related residues to the catalytic activity of protein. Molecular dynamics (MD) simulations can provide guidance for protein engineering. Elimination of hydrogen-bonding forces releases a larger nucleotide of freedom, which improves the catalytic activity of ZaTdT. In this cycle, we obtained two mutants, ZaTdT-K337A and ZaTdT-K337L, that could extend all four 3’-O-blocked reversible terminators, after performing the reduce the frequency of hydrogen-bond formation between the triphosphate group and Lys337. Ultimately, we found that BBa_K5392000 was new basic part because it had better catalytic activity for natural dNTP than other sources of TdTs and BBa_K5392021 was new composite part, because they obtained better catalytic activity compared to wild-type ZaTdT or other ZaTdT mutant for 3’-O-(2-nitrobenzyl)-dNTP that is a 3’-O-blocked reversible terminator.

4. Reference

[1] BOLLUM FJ. Thermal conversion of nonpriming deoxyribonucleic acid to primer. J Biol Chem. 1959;234:2733-2734.

[2] Bollum FJ. Chemically Defined Templates and Initiators for Deoxypolynucleotide Synthesis. Science. 1964;144(3618):560.

[3] Anavy L, Vaknin I, Atar O, Amit R, Yakhini Z. Data storage in DNA with fewer synthesis cycles using composite DNA letters. Nat Biotechnol. 2019;37(10):1229-1236.

[4] Hutchison CA 3rd, Chuang RY, Noskov VN, et al. Design and synthesis of a minimal bacterial genome. Science. 2016;351(6280):aad6253.

[5] Mercy G, Mozziconacci J, Scolari VF, et al. 3D organization of synthetic and scrambled chromosomes. Science. 2017;355(6329):eaaf4597.

[6] Minhaz Ud-Dean SM. A theoretical model for template-free synthesis of long DNA sequence. Syst Synth Biol. 2008;2(3-4):67-73.

[7] Chen F, Dong M, Ge M, et al. The history and advances of reversible terminators used in new generations of sequencing technology. Genomics Proteomics Bioinformatics. 2013;11(1):34-40.

[8] Loc'h J, Rosario S, Delarue M. Structural Basis for a New Templated Activity by Terminal Deoxynucleotidyl Transferase: Implications for V(D)J Recombination. Structure. 2016;24(9):1452-1463.

[9] Delarue M, Boulé JB, Lescar J, et al. Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase. EMBO J. 2002;21(3):427-439.

[10] Sosunov VV, Santamaria F, Victorova LS, Gosselin G, Rayner B, Krayevsky AA. Stereochemical control of DNA biosynthesis. Nucleic Acids Res. 2000;28(5):1170-1175.

[11] Guo J, Yu L, Turro NJ, Ju J. An integrated system for DNA sequencing by synthesis using novel nucleotide analogues. Acc Chem Res. 2010;43(4):551-563.

[12] Gouge J, Rosario S, Romain F, Beguin P, Delarue M. Structures of intermediates along the catalytic cycle of terminal deoxynucleotidyltransferase: dynamical aspects of the two-metal ion mechanism. J Mol Biol. 2013;425(22):4334-4352.

[13] X Lu, J Li, C Li, et al. Enzymatic DNA Synthesis by Engineering Terminal Deoxynucleotidyl Transferase. ACS Catalysis. 2022; 12(5):2988-2997.