Terminal deoxyribonucleotidyl transferase mutants, their preparation methods, and reagent kits

Abstract

This invention belongs to the field of biosynthesis technology and relates to a terminal deoxyribonucleotidyl transferase mutant, its preparation method, and a reagent kit. The mutant is obtained by multi-point mutation of the truncated wild-type terminal deoxyribonucleotidyl transferase amino acid sequence, with mutation sites including R336L, K338G, L397M, and E456S or E456G. Compared with existing technologies, this invention achieves rational modification of wild-type BtTdT in a shorter time, with higher modification efficiency, and the resulting mutant exhibits significantly improved catalytic efficiency, representing the best reported activity for catalyzing 3'-ONH2-dNTPs. Furthermore, the modification method of this mutant can be applied to modify other TdTs to improve the catalytic efficiency of TdTs for other 3'-block substrates.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to terminal deoxyribonucleotidyl transferase mutants, their preparation methods, and reagent kits. Background Technology

[0002] The vast majority of biological research relies on DNA synthesis, including primer synthesis, gene synthesis, and even the synthesis of entire chromosomes. Furthermore, DNA synthesis holds significant promise for applications in information storage. With the explosive growth of information, current silicon-based information storage technologies are insufficient to meet the demands of information storage due to their energy-intensive processes and limited storage capacity. DNA, on the other hand, is stable, with a half-life of approximately 521 years, requires less energy for storage, and has a data storage capacity several orders of magnitude greater than silicon-based materials. Numerous researchers are dedicated to developing next-generation DNA-based information storage technologies. In this technology, DNA synthesis is an indispensable and crucial component.

[0003] Currently, DNA synthesis mainly relies on the mature chemical solid-phase synthesis method, namely the phosphoramide process. Developed in 1981, this method, despite continuous optimization, can only synthesize DNA sequences up to 200-300 base pairs in length. To synthesize longer DNA sequences, DNA assembly techniques are required. Solid-phase synthesis also has other drawbacks, such as the need for large quantities of toxic and harmful chemical reagents and the resulting wastewater posing environmental hazards.

[0004] Enzymatic DNA synthesis technology can overcome the shortcomings of chemical methods and has become a very promising alternative. Enzymatic DNA synthesis mainly relies on terminal deoxynucleotidyltransferase (TdT), an enzyme first discovered in the early 1960s. It is a template-free DNA polymerase that can indiscriminately add dNTPs to the 3' end of ssDNA, thereby continuously extending the DNA strand. In recent years, studies have successfully synthesized DNA sequences of 10 or 50 nucleotides in length using TdT, making it possible to synthesize even longer DNA sequences.

[0005] The most promising application of TdT enzymatic DNA synthesis currently lies in using 3'-OH protected reversibly terminated dNTPs (RTdNTPs) as extension units to synthesize DNA by synthesizing only one base per cycle. However, the natural substrate of wild-type TdT is dNTP, and its catalytic activity against non-natural substrates RTdNTP is low and the conversion is incomplete. This severely limits the development and industrial application of TdT DNA synthesis processes. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention rationally modifies wild-type BtTdT by point mutation to obtain a mutant of BtTdT. The modified BtTdT mutant has a significantly improved catalytic efficiency for the non-natural substrate RTdNTP compared to the wild-type enzyme.

[0007] One of the terminal deoxyribonucleotidyl transferase mutants provided by the present invention is obtained by point mutations of R336L, K338G and L397M on the truncated wild-type terminal deoxyribonucleotidyl transferase amino acid sequence, and its amino acid sequence is shown in SEQ ID NO.1 of the sequence listing.

[0008] Another terminal deoxyribonucleotidyl transferase mutant provided by the present invention is obtained by performing an E456S or E456G point mutation on the amino acid sequence shown in SEQ ID NO. 1, and its amino acid sequence is shown in SEQ ID NO. 2 or 3.

[0009] The present invention further provides a method for preparing the terminal deoxyribonucleotidase mutant, comprising the following steps:

[0010] (1) The amino acid sequence of the wild-type terminal deoxyribonucleotidyl transferase was shortened and the BRCT domain was removed;

[0011] (2) Perform three-point mutations on the truncated amino acid sequence to obtain a mutant with the sequence shown in SEQ ID NO.1 of the sequence listing; or perform four-point mutations to obtain a mutant with the amino acid sequence shown in SEQ ID NO.2 or 3 of the sequence listing.

[0012] Preferably, the three point mutations include R336L, K338G, and L397M.

[0013] Preferably, the four-site mutations include R336L, K338G, L397M, and E456S; or R336L, K338G, L397M, and E456G.

[0014] Preferably, the mutant containing E456S is obtained by structural prediction using a protein structure prediction tool.

[0015] The present invention also provides a nucleic acid that encodes the terminal deoxyribonucleotidase mutant.

[0016] The present invention also provides a kit comprising the nucleic acid or the terminal deoxyribonucleotidase mutant described herein.

[0017] The beneficial effects of this invention are: compared with the prior art, this invention takes less time to rationally modify BtTdT, has higher modification efficiency, and the catalytic efficiency of the resulting mutant is significantly improved, making it the best reported mutant for catalytic 3'-ONH2-dNTPs; moreover, the modification method of this mutant can be applied to modify other TdTs to improve the catalytic efficiency of TdTs for other 3'-block substrates. Attached Figure Description

[0018] Figure 1 The study included the structure of wild-type BtTdT and its truncated activity assays, including: a. the two main domains of wild-type BtTdT; b. the expression of BtTdT with its N-terminus truncated (BRCT domain removed) with the his tag; and c. the activity assays of N-terminal truncated BtTdT, with the ordinate representing relative activity and the abscissa representing the number of amino acids elongated at the N-terminus of the truncated POLXc domain.

[0019] Figure 2 Structural prediction and amino acid residue comparison of BtTdT and ZaTdT, and activity assay of BtTdT mutants; including: a. The 397 site of BtTdT (green) or BtL397M (magenta); L397 hinders base stacking between the second and third nucleotides at the 3' end of the primer, while the L397M mutation alleviates the inhibition of base stacking; b. Expression of BtL397M; c. Comparison of key amino acids of R336L / K338G in Bt15AA-R336L-K338G (M2, green) and ZaTdT-R335L-K337G (cyan); d. Expression of BtTdT mutants; e. Extension efficiency of Bt15AA-R336L-K338G-L397M (denoted as M3) with different primers; f. Activity test of BtL397M; g. Comparison of the activities of BtTdT mutants;

[0020] Figure 3 The study compared the differential amino acids within 5 Å of the active sites of mutants M3 and ZaTdT-R336L-K338G, as well as their activities after mutation. Specifically: a. Differential amino acids within 5 Å of the active sites of M3 (green) and ZaTdT-R336L-K338G (cyan); b. Single mutations and combination mutations of T396A / G or E456A / G, and their activity assays based on the M3 mutant; c. Elongation of M3-E456G with 16 primers and 3'-ONH2-dATP; d. Comparison of the elongation efficiency of BtTdT wild-type and mutants with 3'-block-dGTP containing blocking groups of different lengths.

[0021] Figure 4The study compared the structure and activity of the enzyme after the E456 amino acid mutation. Specifically: a. The change in the distance between the 3'-OH group of substrate ATP and the enzyme surface when the E456 amino acid was mutated on the M3 base: E456S (yellow), E456G (cyan), E456 (green), and E456A (magenta); b. Activity assay of the M4 mutant with added 3'-ONH2-dATP under different time conditions; c. 3'-ONH2-dNTP was added to M4 within 5 min. Detailed Implementation

[0022] The present invention will now be described more completely and clearly with reference to the accompanying drawings and specific embodiments. The described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0023] This invention provides mutants obtained by modifying wild-type BtTdT enzyme (amino acid sequence as shown in SEQ ID NO.4 of the sequence listing), wherein the amino acid sequences of the three-point mutants R336L, K338G, and L397M are shown in SEQ ID NO.1 of the sequence listing; and the amino acid sequences of mutants with the addition of E456S or E456G are shown in SEQ ID NO.2 or 3 of the sequence listing, respectively.

[0024] The process of modifying wild-type BtTdT enzyme to obtain mutants in this invention is as follows:

[0025] 1. Truncation of wild-type TdT enzyme

[0026] The complete structure of wild-type TdT enzymes can be divided into two domains, such as Figure 1 As shown in Figure a, the BRCT domain and POLXc domain are respectively. The BRCT domain is not related to the catalytic activity of TdT, while the core domain containing the POLXc domain has TdT activity. This invention constructed TdT enzymes of different truncated lengths (with the BRCT domain removed) and investigated the expression and catalytic activity of different truncated mutants. The mutants of different lengths were expressed, purified, and their catalytic activity against the substrate dNTPs was analyzed. It was found that the full-length TdT enzyme had lower catalytic activity than the truncated mutants. Furthermore, the highest activity was achieved when the original 15 amino acid length was extended at the N-terminus of POLXc. When the number of amino acids extended at the N-terminus was less than 5, the soluble expression level was extremely low when expressed as a His6-tagged fusion protein in *E. coli*, and it mainly existed in the form of inclusion bodies. Figure 1 As shown in b and c.

[0027] 2. Amino acid residue mutation of BtTdT

[0028] Before performing protein engineering, a reliable three-dimensional structure of TdT is required. Therefore, this invention employs the commonly used prediction method AlphaFold to generate its accurate three-dimensional structure. Because the L398M mutation increases the distance between the TdT and the 3' terminal nucleotide bases of the primer, it can alleviate the inhibition of the amino acid at this site on the stacking interaction of the primer's 3' terminal nucleotide bases to some extent, thereby improving the enzyme reaction rate, such as... Figure 2 As shown in Figure a. Experimental results show that the L397M mutant exhibits approximately twice the catalytic efficiency for 3'-ONH2-dNTPs compared to the wild-type BtTdT, such as... Figure 2 As shown in b and f.

[0029] Furthermore, previous reports have shown that the combined mutation R336L / K338G is beneficial for improving the enzyme's catalytic activity towards the substrate 3'-ONH2-dNTPs. Therefore, this invention also compares the structures of Bt15AA-R336L-K338G (denoted as M2) and ZaTdT-R335L-K337G. Since the spatial positions of the corresponding residues R336L and K338G after the mutations are almost completely overlapping, these two residue mutations in BtTdT are likely to contribute to improving its conversion rate of 3'-ONH2-dNTPs, such as... Figure 2 As shown in Figure c. The results show that the activity of the double mutant M2 is 5 times stronger than that of the wild-type BtTdT, which well verifies the hypothesis of this invention. The three-site combination mutants of R336L, K338G, and L397M further improved the catalytic activity of 3'-ONH2-dTTP by about 60%, as shown in Figure c. Figure 2 As shown in d and g. Subsequently, this invention tested the catalytic efficiency of the three-point mutant Bt15AA-R336L-K338G-L397M (denoted as M3) on 16 primers (4×4) terminated with different dinucleotides and 4 3'-ONH2-dNTP substrates. The catalytic activity was greatly improved. Except for the lower conversion rate when 3'-ONH2-dATP was added to primers terminated with TC or CC, all other substrate combinations could be almost completely converted within 10 min. Figure 2 As shown in Figure e. Because this three-point mutant M3 cannot completely transform primers terminated by TC or CC within 10 min, its activity is not high enough and needs further modification to be suitable for efficient enzymatic DNA synthesis.

[0030] Since ZaTdT-R336L-K338G exhibits complete extension of 3'-ONH2-dATP for primers ending in TC, this invention compares the key amino acid residues within 5 Å of the catalytic center of ZaTdT-R336L-K338G and mutant M3 to further enhance the catalytic activity of the BtTdT mutant for the addition of 3'-ONH2-dATP. Figure 3 As shown in Figure a. Clearly, T396 and E456 in mutant M3 are the different amino acids between the two, which are replaced by A and G, respectively, in ZaTdT-R336L-K338G. This invention hypothesizes that these two smaller residue substitutions may provide greater activity space for 3'-ONH2-dATP; therefore, T396G / A and E456A / G mutants were constructed based on M3 to test their effect on the catalytic efficiency of 3'-ONH2-dATP. All four mutants showed higher conversion rates than the parental mutant M3, especially the E456G mutant, which showed the best catalytic effect, completely converting primers terminated by TC and CC within 10 and 20 min, respectively. Figure 3 As shown in b. Further combinatorial mutations were then performed on the E456G base with T396G / A. However, compared to the four-point mutant M3-E456G, the five-point mutant did not further enhance catalytic activity, as shown in Figure b. Figure 3 As shown in Figure b. The efficiency of M3-E456G in adding 3'-ONH2-dATP to 16 primer pairs was also determined, confirming that the increased activity did not reduce its catalytic ability for other substrates, such as... Figure 3 As shown in c. The results indicate that only primers ending in CC could not be completely transformed within 10 minutes. This "weakest link" needs further improvement through enzyme engineering to obtain a better mutant.

[0031] 3. Rational Design Based on the Protein Prediction Tool AlphaFold 3 (AF3)

[0032] Considering that site E456 is crucial for the catalytic efficiency of 3'-block dNTPs, increasing the pocket size to provide greater freedom for nucleotides can improve catalytic efficiency. Furthermore, this invention suggests that a more suitable amino acid substitution may exist at site E456, which would increase the distance between the 3'-ONH2 group and the enzyme surface, thereby granting the substrate greater freedom and enabling it to form more favorable non-covalent bonds such as hydrogen bonds with surrounding amino acids, thus achieving higher catalytic activity. Simultaneously, to reduce workload and shorten research time, this invention uses the state-of-the-art protein structure prediction tool AF3 to predict the structure of mutants with 20 amino acids mutated at site E456 in M3. Since AF3 can provide ligands such as metal ions or DNA strands during docking, ATP, the substitute substrate for 3'-ONH2-dATP, was chosen as the ligand during prediction. Modeling results show that only in the E456S mutant is the distance between the 3'-OH group of the substrate mononucleotide and the enzyme surface the greatest, suggesting that the E456S mutation may further increase the enzyme's catalytic activity, such as... Figure 4 As shown in Figure a. The M3-E456S (denoted as M4) mutant was expressed and purified, and its elongation efficiency for 3'-ONH2-dNTPs was then tested. The results showed that mutant M4 could achieve complete substrate transformation and shorten the reaction time to 5 minutes, as shown in Figure a. Figure 4 As shown in Figure b, when the extension time of 1 min was tested with 3'-ONH2-dATP, none of the three primers (terminated with TC, CT, or CC) could completely convert the substrates. Furthermore, the catalytic activity of M4 for three other 3'-ONH2-dNTPs was also tested, and the results showed that all 64 substrate pairs could be completely converted within 5 minutes. Figure 4 As shown in c.

Claims

1. A terminal deoxyribonucleotidyl transferase mutant, characterized in that: The mutant was obtained by point mutations of R336L, K338G, and L397M into the truncated wild-type terminal deoxyribonucleotidyl transferase amino acid sequence, and its amino acid sequence is shown in SEQ ID NO.1 of the sequence listing.

2. The terminal deoxyribonucleotidase mutant according to claim 1, characterized in that: The mutants are obtained by performing point mutations of E456S or E456G on the amino acid sequence shown in SEQ ID NO.1, and their amino acid sequences are shown in SEQ ID NO.2 or 3, respectively.

3. The method for preparing the terminal deoxyribonucleotidyl transferase mutant as described in claim 1 or 2, characterized in that, Includes the following steps: (1) The amino acid sequence of the wild-type terminal deoxyribonucleotidase was truncated and the BRCT domain was removed; (2) Perform three-point mutations on the truncated amino acid sequence to obtain a mutant with an amino acid sequence as shown in SEQ ID NO.1 of the sequence listing; or perform four-point mutations to obtain a mutant with an amino acid sequence as shown in SEQ ID NO.2 or 3 of the sequence listing.

4. The method for preparing the terminal deoxyribonucleotidyl transferase mutant according to claim 3, characterized in that, The three point mutations mentioned include R336L, K338G, and L397M.

5. The method for preparing the terminal deoxyribonucleotidyl transferase mutant according to claim 3, characterized in that, The four-site mutations include R336L, K338G, L397M, and E456S; or R336L, K338G, L397M, and E456G.

6. The method for preparing the terminal deoxyribonucleotidyl transferase mutant according to claim 5, characterized in that, The mutant containing E456S was obtained by structural prediction using a protein structure prediction tool.

7. A nucleic acid, characterized in that, The nucleic acid encodes the terminal deoxyribonucleotidase mutant as described in claim 1 or 2.

8. A reagent kit, characterized in that, It includes the nucleic acid as described in claim 7.

9. A reagent kit, characterized in that, It includes the terminal deoxyribonucleotidase mutant as described in claim 1 or 2.

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