Latest revision as of 08:53, 2 October 2024

Thermostable Terminal deoxynucleotidyl Transferase (ThTdT)

Thermostable Terminal Deoxynucleotidyl Transferase (ThTdT) is a mutated template-independent DNA polymerase originating from Bos taurus. This protein coding basic part performs template-free nucleotide triphosphate (dNTP) addition at the 3’ end, requiring a starting primer. Recombinantly isolated from DH5α E. coli, the enzyme is a thermostable variant of its wild type counterpart enabling an increased working temperature range of 37°C to 55°C (as tested) and requires a divalent cation such as Co²⁺ to increase its 3’-extension efficiency (used in characterization experiments).

Sequence and Features

1. Usage and Biology

1.1 Terminal deoxynucleotidyl Transferase (TdT)

1.1.1 Wild type TdT (WT TdT) Overview

Terminal deoxynucleotidyl transferase (TdT) is a uniquely template-independent DNA polymerase (Ashley et al., 2023). Chemically, TdT elongates the free 3’-hydroxyl termini of DNA molecules, typically a primer, using deoxyribonucleotide triphosphates (dNTPs) as substrates, forming inorganic pyrophosphate (PP_i) as a by-product. It plays a key role in diversifying the human cell receptor portfolio of T- and B- cell receptors by V(D)J recombination. This is achieved by adding non-templated nucleotides between the V, D, J exons (Motea & Berdis, 2010).

TdT belongs to the X family of polymerases, responsible for DNA repair in mammals (Hoitsma et al., 2020). It was one of the first mammalian polymerases to be identified, originally extracted from the cow thymus in 1960 (Bollum, 1960). Therefore, the wild type TdT (WT TdT) we use for our project (see below), nuCloud, comes from cows. Similarly, the thermostable TdT (ThTdT) used is also of bovine origin (Chua et al., 2020).

As a transferase (Enzyme Commission EC 2.7.7.31), TdT catalyzes the following reaction:

Oligonucleotide_n + dNTP → Oligonucleotide_n+1 + PP_i

WT TdT has optimal activity at approximately 37ºC, and it inactivates at 40ºC (Chua et al., 2020; Boulé et al., 2000). TdT is also a metalloenzyme, where either one of Mg²⁺ or Mn²⁺ is required for catalytic activity (Pandey et al., 1987). Other divalent transition metal cations, such as Co²⁺ and Zn²⁺ are known to enhance its transferase activity, especially with dCTP and dTTP (Grosse et al., 1993; Ratliff, 1981).

1.1.2 Thermostable TdT (ThTdT) Overview

As TdT adds nucleotides to the growing ssDNA strand, the increasing sequence length reduces the energetic stability of the ssDNA (Bochman et al., 2012). This causes the oligonucleotide strand to fold back on itself, forming undesired secondary structures. These secondary structures, in particular, have been shown to reduce the efficiency of TdT activity (Barthel et al., 2020).

To address the incumbrance of secondary structures, increasing the reaction temperature is a preferable approach, as it weakens intramolecular hydrogen bonds and prevents the formation of secondary structures (Barthel et al., 2020). To facilitate the reaction at elevated temperatures, we engineered a more thermostable version of TdT using synthetic biology techniques, which we named thermostable TdT (ThTdT). ThTdT is designed to retain functionality at elevated temperatures, enabling reliable long oligonucleotide production without secondary structure interference. According to Barthel et al. (2020), ThTdT was shown to function up to 47ºC, 10 ºC higher than the unmodified TdT.

ThTdT carries 11 point mutations relative to the wildtype, where 9 are outside of the substrate binding pocket. Within the binding pocket, two substitution mutations were found in the active site, M339K and T340I. Notably, M339K was predict to afford a new hydrogen bond with G337, which may confer higher thermostability alongside with other mutations. While this conformer requires further validation, other possible stabilization modes, such as solvation, may be plausible in the improved thermostability.

1.2 Current Applications of TdT

Beyond applications in DNA data storage, TdT is used in other areas of research and industry:

• Research

  • Molecular probe generation
    • Using chemically modified nucleotide triphosphates, such as fluorophore and digoxigenin conjugates, antisense based molecular probes were synthesized by TdT to generate highly specific targeting agents for biological imaging and disease etiology studies (Lu et al., 2022).
  • Polynucleotide synthesis
    • Standard DNA synthesis uses phosphoramidite chemistry and require punctiliously anhydrous and volatile organic reagents, as opposed to chemoenzymatic synthesis that occurs in aqueously buffered salts (Barthel et. al, 2020). Pivoting to enzymatic synthesis via optimized use of polymerases, like TdT, provides scope for cheaper and greener polynucleotide synthesis.
  • DNA nanotechnology
    • TdT has been demonstrated to aid in the assembly of DNA nanostructures such as hydrogels. DNA hydrogels require DNA building blocks, which can be enzymatically synthesized using TdT (Xiang et. al, 2016). TdT can be used to add complementary DNA tails to the building blocks which allows for hybridization between the blocks and formation of the gel structure.
  • Biosensors
    • TdT has been applied to develop new biosensing platforms as well as to amplify the output of biosensors. TdT’s ability to add multiple nucleotides of the same base can be used to generate poly-dATP tails. These polynucleotide tails have been used for sensitive detection of the activity of various molecules such as uracil-DNA glycosylase (Du et. al, 2021).

• Industry

  • Companies have also started adopting the use of TdT in developing new products to perform DNA synthesis. DNAScript launched the first benchtop DNA printer, SYNTAX, run on enzymatic DNA synthesis technology, and TdT plays a critical role in the elongation of DNA in the workflow (Grinstein, 2023).

1.3 Usage with nuCloud

1.3.1 About nuCloud

Our project, nuCloud, aims to address the need for a sustainable, more energy-efficient data storage medium compared to current magnetic and optical data storage options. One direction through which we tackle this issue is by developing an enzymatic DNA synthesis platform that can elongate single-stranded DNA (ssDNA) in a template-independent manner. The synthesized ssDNA strand will then be converted to a more stable, double-stranded DNA (dsDNA) and can be inserted into a plasmid for long-term data storage. We will leverage TdT’s ability to elongate DNA in a template-independent manner to perform enzymatic synthesis of the original ssDNA strands that are needed for data storage.

1.3.2 ThTdT in nuCloud

In nuCloud, ThTdT enables the synthesis of custom nucleotide sequences encoding data for storage efficiently and sustainably. Even with the efficiency gains of ThTdT, template-free DNA synthesis remains a highly repetitive process, adding only one nucleotide per reaction cycle. To overcome this, we leverage microfluidic platforms to automate the synthesis process and minimize the amount of ThTdT and reagents used.

Prior to solid-phase DNA synthesis, where the primer sequence is immobilized to a glass slide, we tested our ThTdT in a conventional liquid-phase system to verify its ability to add nucleotides to the primer. Liquid-phase synthesis refers that all components - the ThTdT enzyme, deoxyribonucleotide triphosphates (dNTPs), primer, and Co²⁺ - are in solution. The simple setup also allowed us to perform characterization tests on ThTdT over a range of temperatures and concentrations.

Solid-phase DNA synthesis is the strategy that immobilizes the primer to be extended onto a solid support. Unlike LPS, an immobilized primer means that after a nucleotide has been added, the liquid phase can be washed away and a new solution containing the next nucleotide can be added, akin to solid phase oligonucleotide synthesis without the harmful chemicals. This strategy allows us to control which bases are added per cycle which enables us to synthesize any DNA sequence suitable for storing data. We designed and produced reusable microfluidic chips to facilitate solid-phase enzymatic DNA synthesis with our ThTdT, enabling resource-saving small reaction volumes, and an automated workflow.

2. Characterization of ThTdT

2.1 Designing the ThTdT Gene Fragment

To generate the Thermostable TdT gene fragment (ThTdT), the DNA sequence from Chua et al. (2020) was used to combine with an N-terminal His₆, thrombin cleavage site, and SUMO tag in silico. (Fig 2.1).

2.2 Cloning of ThTdT

The pET-28b plasmid backbone was obtained from Novagen. To remove the multiple cloning site (MCS) and undesirably positioned His₆-tag from pET-28b, inverse PCR was performed on pET-28b plasmid. A 30 bp overhang in the ThTdT gene fragment was included in preparation for Gibson assembly (Figure 2.2).

Using Gibson assembly, ThTdT was ligated with the modified pET-28b plasmid. The product was then transformed into chemically competent DH5α E. coli (Figure 2.4). Successful ligation and transformation were indicated via colony growth on an LB-Kanamycin (50 µg/mL) plate. Further validation was completed by colony PCR (Figure 2.5) and whole plasmid sequencing by Plasmidsaurus (Figure 2.6).

2.3 Purification of ThTdT

Plasmids were extracted from DH5α and transformed into BL21 (DE3) for ThTdT protein expression (Fig 2.7). ThTdT expression was induced with IPTG in transformed BL21, followed by mechanical lysis to obtain crude cell lysate. Isolation of ThTdT was completed with Ni-NTA magnetic beads (New England Biolabs, NEB). Fractions containing ThTdT was confirmed via SDS-PAGE (Fig 2.8). To remove the excess imidazole from the protein elution buffer, the combined ThTdT-containing fractions were purified using Amicon® Ultra Centrifugal Filter system to concentrate the samples. For long-term storage, the purified protein was formulated in NEB storage buffer in-house (50 mM potassium phosphate, 100 mM NaCl, 1.43 mM β-mercaptoethanol, 50% Glycerol, 0.1% Triton® X-100, pH 7.3 @ 25°C) that was recreated in-house. The storage buffer was also supplemented with 100 µM 2,2’-bipyridyl to remove residual contaminating Ni²⁺ that was carried over from purification.

2.4 Proof of Function: Liquid Phase Synthesis (LPS)

2.4.1 Benchmark Establishment using WT TdT (LPS)

Using the in-house ThTdT, 3’-elongation activity to Cy5-labeled primer P1 was observed using all four canonical nucleotide triphosphates at temperatures above 40ºC. This was chosen because it was the upper temperature limit for WT TdT to retain its function (Chua et al., 2020). In order to test this hypothesis, a liquid phase synthesis (LPS) system was devised to validate nucleotide addition. Firstly, WT TdT purchased from NEB was studied to develop a standard assaying condition for all four nucleotide triphosphates (Figure 2.9).

This supports the claim that our system was successful in catalyzing nucleotide addition and this condition could be used as a benchmark against our in-house ThTdT. This also validated that denaturing PAGE following fluorescent detection on a Typhoon biomolecular scanner was an efficacious workflow to visualize the results.

2.4.2 Functional Validation of ThTdT (LPS)

With a benchmark assay available, the ability for our in-house ThTdT to incorporate nucleotide triphosphates to an oligonucleotide was studied. Purified ThTdT stored in a modified NEB storage buffer was successfully able to incorporate nucleotides in primer 2.

2.5 Proof of Function: Solid Phase Synthesis (SPS)

2.5.1 Oligonucleotide Functionalization on Glass (SPS)

Encouraged by the success in LPS, the focus turned to solid phase synthesis (SPS). SPS refers to the strategy where an immobilized, yet cleavable, oligonucleotide to be 3’-extended using any TdT. The reason why a base-cleavable linker was included was to allow rapid cleavage of the ThTdT-treated ssDNA for denaturing PAGE analysis. To encapsulate these notions, P1 was designed for this purpose.

A few steps were applied to a microscope glass slide in order to receive the primer for functionalization (Fig. 2.12). The slides were first silanized using Piranha solution (25% v/v H₂O₂, 75% v/v H₂SO₄), followed by introduction of 3-aminopropytriethyoxysilane (APTES) to install free amine groups. Following standard amide coupling conditions with N-hydroxysuccinimide esters of PEGylated acids and biotin conjugates (99:1 mole ratio), a biotinylated glass surface was afforded. Since streptavidin is a potent binder of biotin, it serves as a natural linker between two biotinylated moieties, namely the biotinylated glass slide and the biotinylated P1.

2.5.2 Functional Validation of ThTdT (SPS)

Via fluorescent imaging, Figure 2.13 demonstrated that P1 was functionalized onto glass. ThTdT, along with other cofactors and substrates, were directly applied to the oligonucleotide-immobilized glass as a 10 µL droplet.

The reaction droplets were removed by pipetting, followed by aqueous washes to remove residual reactants. Gel loading buffer, supplemented with 0.1 M NaOH, was loaded directly on top of the reacted glass patches to quench the reaction and to cleave and denature the ssDNA of interest for separation on denaturing PAGE.

This SPS system demonstrated promising preliminary efficacy in incorporating dTTP to P1, as demonstrated by Figure 2.14 where the SPS-treated sample had more mass than the primer-only negative control following cleavage in NaOH.

3. References

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• Barthel, S., Palluk, S., Hillson, N. J., Keasling, J. D., & Arlow, D. H. (2020). Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis. Genes, 11(1), 102. https://doi.org/10.3390/genes11010102
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