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WO2025140014A1 - Utilisation d'un mutant de désoxynucléotidyl transférase terminale dans la synthèse d'acides nucléiques - Google Patents

Utilisation d'un mutant de désoxynucléotidyl transférase terminale dans la synthèse d'acides nucléiques Download PDF

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Publication number
WO2025140014A1
WO2025140014A1 PCT/CN2024/140779 CN2024140779W WO2025140014A1 WO 2025140014 A1 WO2025140014 A1 WO 2025140014A1 CN 2024140779 W CN2024140779 W CN 2024140779W WO 2025140014 A1 WO2025140014 A1 WO 2025140014A1
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sequence
solid phase
nucleic acid
optionally
active substance
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PCT/CN2024/140779
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Chinese (zh)
Inventor
高南风
杨卫康
于爱淼
谢庆庆
沈玥
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Bgi Research Changzhou
Gcatbio Co Ltd
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Bgi Research Changzhou
Gcatbio Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the present application relates to the field of biotechnology, and in particular to the application of a terminal deoxynucleotidyl transferase mutant in nucleic acid synthesis.
  • Phosphoramidite synthesis is the mainstream method for synthesizing short-chain DNA, which can reliably provide short DNA chains of ⁇ 200 nucleotides.
  • the phosphoramidite synthesis method mainly completes a single base addition cycle through four steps of deprotection, coupling, capping and oxidation. The steps are cumbersome, and there are problems such as a long single cycle time (6-8 minutes), high consumption of chemical reagents, and high cost.
  • Terminal deoxynucleotidyl transferase is a DNA polymerase that is widely used and has obvious advantages in enzymatic de novo DNA synthesis technology.
  • TdT can indiscriminately extend four natural nucleotides (A, T, C, G) to the 3' end of the starting chain without a template.
  • A, T, C, G natural nucleotides
  • CN114921436A discloses a terminal deoxynucleotidyl transferase mutant with improved thermal stability, which mutates the sites of the amino acid sequence of the wild-type terminal deoxynucleotidyl transferase shown in SEQ ID NO.1 to obtain a mutant with six mutations of N135P, S138H, Q229D, K232D, L234R, and V366M.
  • the embodiments of the present application provide a terminal deoxynucleotidyl transferase mutant and a preparation method and application thereof, in order to solve the problems of low efficiency, reaction energy consumption, high pollution, etc. in synthesizing DNA by the traditional phosphoramidite synthesis method, as well as the low catalytic efficiency of wild terminal deoxynucleotidyl transferase.
  • the present application provides a terminal deoxynucleotidyl transferase mutant, wherein the amino acid sequence of the terminal deoxynucleotidyl transferase mutant comprises any one of the following sequences:
  • the following mutations occur based on the sequence shown in SEQ ID NO:2: any one or at least two of the following sequences: E459R, E459K, E459Q, E459V, R460Q, R460K, R460E, R460H, R460N, R460D, R460S, R460L, R460M, R460W, R460F, R460Y, R460C or R460G; or
  • FIG. 3 is a Urea-PAGE diagram of the terminal deoxynucleotidyl transferase product according to an example of the present application.
  • FIG. 4 is a capillary electrophoresis diagram of transformation products of GeTdT wild type and its E459R and E459R/R460Q mutants according to an embodiment of the present application.
  • FIG6 shows a process of solid phase nucleic acid synthesis based on enzymatic reaction according to an embodiment of the present application.
  • FIG7 shows the product detection results of solid phase nucleic acid synthesis based on enzymatic reaction according to an embodiment of the present application
  • FIG8 shows a modified nucleotide with a protecting group that can be used for solid phase synthesis according to an embodiment of the present application.
  • FIG. 9 shows the polynucleotide sequences synthesized under the catalysis of GeTdT mutants with or without protease treatment according to an embodiment of the present application.
  • the nucleotide sequence is shown in SEQ ID NO.1 and the amino acid sequence is shown in SEQ ID As shown in NO.2, primers containing E459R, E459K, E459Q, E459V, E459R/("/" indicates sum)R460Q, E459R/R460K, E459R/R460E, E459R/R460H, E459R/R460N, E459R/R460D, E459R/R460S, E459R/R460L, E459R/R460M, E459R/R460W, E459R/R460F, E459R/R460Y, E459R/R460C, and E459R/R460G mutations were designed and synthesized according to the sequence, and site-directed mutagenesis of GeTdT was performed.
  • the recombinant expression plasmid pGS-21a/GeTdT (G144—A512) carrying the gene encoding the partial peptide chain of GeTdT (amino acids G144 to A512 in SEQ ID NO.2, i.e., the wild-type GeTdT described later) as a template (the complete peptide chain sequence expressed is shown in SEQ ID NO.3), mutations were introduced at characteristic sites by rapid PCR technology using the corresponding mutation primer pairs shown in Table 1, and Sanger sequencing was used to confirm whether the encoding gene of the GeTdT mutant was correct.
  • the PCR reaction system was: 10 ⁇ L of 5 ⁇ PS buffer, 4 ⁇ L of dNTPs Mix (2.5 mmol/L), 1 ⁇ L of forward primer (10 ⁇ mol/L), 1 ⁇ L of reverse primer (10 ⁇ mol/L), 1 ⁇ L of template DNA, 0.5 ⁇ L of Primer Star HS (Takara, 5 U/ ⁇ L), and distilled water was added to 50 ⁇ L.
  • the PCR amplification program was set as follows: first, pre-denaturation at 94°C for 5 min; then 21 cycles (denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 7 min 50 s); finally, extension at 72°C for 7 min, and insulation at 4°C. PCR products were detected by 1% agarose gel electrophoresis.
  • Dpn I was added to the PCR product verified by gel electrophoresis, and the template was degraded by water bath at 37°C for 2 hours, and then transformed into Escherichia coli JM109 competent cells (purchased from Tiangen Biochemical Technology Co., Ltd.).
  • the transformation product was spread on LB solid medium containing 100 mg/L ampicillin, cultured at 37°C for 11 hours, and the clones were picked and inoculated into LB liquid medium and cultured at 37°C for 9 hours.
  • the bacterial solution was subjected to Sanger sequencing to verify whether the mutation site was correctly introduced.
  • the clones with correct sequencing were subjected to plasmid extraction and transformed into the expression host Escherichia coli BL21 (DE3) competent cells (purchased from Tiangen Biochemical Technology Co., Ltd.), and 19 recombinant Escherichia coli strains capable of expressing 18 mutants and GeTdT wild-type proteins were obtained.
  • the 19 recombinant E. coli obtained in Example 1 were inoculated into LB medium respectively, and after culturing at 37°C for 8 hours, they were transferred to 300mL TB fermentation medium at an inoculation amount of 5% of the volume of the fermentation medium. First, they were placed at 37°C and 200rpm for constant temperature culture for 3 hours. When the bacterial OD 600 was 0.5-0.7, IPTG was added to a final concentration of 1 ⁇ M and the culture was induced at 25°C and 200rpm for 24 hours.
  • the fermentation broth was crushed by high-pressure homogenization (crushing conditions were 4°C and 80MPa) and then centrifuged (12000rpm, 30min, 4°C), and the supernatant was the crude enzyme solution of the terminal deoxynucleotidyl transferase mutant produced by the recombinant bacteria.
  • the enzyme solution sample prepared in the previous step was subjected to protein purification, as follows: the terminal deoxynucleotidyl transferase protein was purified by nickel ion affinity chromatography. The crude enzyme solution of the terminal deoxynucleotidyl transferase mutant was loaded onto a nickel ion affinity chromatography column (nickel column) respectively.
  • the recombinant protein was purified by gradient elution method: purification buffer A and purification buffer B were used to prepare eluent 1 (containing 0% buffer B), eluent 2 (containing 5% buffer B), eluent 3 (containing 10% buffer B), eluent 4 (containing 20% buffer B), eluent 5 (containing 50% buffer B) and eluent 6 (containing 100% buffer B), and then the protein bound to the nickel column was gradually eluted with the eluents in the order of imidazole concentration from low to high. During the purification process, all eluents were precooled to 4°C to ensure that the recombinant protein maintained biological activity.
  • the eluent 4 containing the purified target protein was ultrafiltered and concentrated using an ultrafiltration tube that could retain 30 kDa protein, and the purified eluent was replaced with 2 ⁇ enzyme storage buffer. Finally, an equal volume of glycerol was added to the concentrated protein, mixed and stored at -20°C. The purity of the purified recombinant protein was analyzed by SDS-PAGE.
  • M represents protein standard; lanes 1-19 represent: 1, WT (wild type); 2, E459R; 3, E459K; 4, E459Q; 5, E459V; 6, E459R/R460Q; 7, E459R/R460K; 8, E459R/R460E; 9, E459R/R460H; 10, E459R/R460V.
  • 0N 11, E459R/R460D; 12, E459R/R460S; 13, E459R/R460L; 14, E459R/R460M; 15, E459R/R460W; 16, E459R/R460F; 17, E459R/R460Y; 18, E459R/R460C; 19, E459R/R460G. According to Figure 2, it can be seen that the target protein with higher purity was obtained.
  • Purification buffer A (pH 8.0): 20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole.
  • Purification buffer B (pH 8.0): 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole.
  • Oligo(dT) 18 an oligonucleotide substrate, was extended by one base under the action of terminal deoxynucleotidyl transferase to obtain the product Oligo(dT) 18- dNTP-3'-O-Azidomethyl.
  • the reaction system for the activity test of the wild-type GeTdT enzyme and its mutants is shown in Table 2. After the reaction system is mixed, react at 37°C for 10 minutes. After the reaction is completed, the reaction is terminated by heating at 95°C for 10 minutes. Urea polyacrylamide gel electrophoresis (20% denaturing gel) was used to semi-quantitatively analyze the reaction products of GeTdT wild type and its mutants, and the results are shown in Figure 3.
  • the formula of the 5 ⁇ reaction buffer used was: 0.5M Na-Cacodylate, 5mM CoCl 2 , pH 7.2.

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Abstract

L'invention concerne un procédé de synthèse d'acides nucléiques, ledit procédé comprenant : l'utilisation d'un mutant de désoxynucléotidyl transférase terminale pour catalyser la ligature de dNTP à l'extrémité 3'-OH d'un brin unique d'oligodésoxynucléotide, la séquence d'acides aminés du mutant de désoxynucléotidyl transférase terminale comprenant l'une quelconque des séquences suivantes : (1) une séquence présentant une mutation au niveau du ou des sites suivants dans la séquence représentée dans SEQ ID NO : 2 : a. en position 459, ou b. en positions 459 et 460 ; (2) une séquence obtenue par substitution, suppression ou ajout d'un ou d'au moins deux résidus d'acides aminés dans la séquence représentée en (1), présentant une fonction identique ou similaire à la séquence représentée en (1) ; ou (3) une séquence présentant une homologie de séquence d'au moins 90 % avec la séquence représentée en (1) ou en (2), présentant une fonction identique ou similaire à la séquence représentée en (1).
PCT/CN2024/140779 2023-12-26 2024-12-19 Utilisation d'un mutant de désoxynucléotidyl transférase terminale dans la synthèse d'acides nucléiques Pending WO2025140014A1 (fr)

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CNPCT/CN2023/142043 2023-12-26
PCT/CN2023/142043 WO2025137872A1 (fr) 2023-12-26 2023-12-26 Mutant de désoxyribonucléoside transférase terminale, procédé de préparation s'y rapportant et utilisation associée

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PCT/CN2024/140779 Pending WO2025140014A1 (fr) 2023-12-26 2024-12-19 Utilisation d'un mutant de désoxynucléotidyl transférase terminale dans la synthèse d'acides nucléiques

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109477080A (zh) * 2016-06-14 2019-03-15 Dna斯克瑞普特公司 polX家族的DNA聚合酶的变体
US20200263152A1 (en) * 2017-05-22 2020-08-20 The Charles Stark Draper Laboratory, Inc. Modified template-independent dna polymerase
CN112105725A (zh) * 2018-01-08 2020-12-18 Dna斯克瑞普特公司 末端脱氧核苷酸转移酶的变体及其用途
US20210009969A1 (en) * 2014-10-20 2021-01-14 Molecular Assemblies, Inc. Modified template-independent enzymes for polydeoxynucleotide synthesis
CN112746063A (zh) * 2019-10-29 2021-05-04 中国科学院天津工业生物技术研究所 核苷转移酶的新功能及应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116555216A (zh) * 2022-01-28 2023-08-08 中国科学院天津工业生物技术研究所 可控合成单链dna的末端转移酶变体及应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210009969A1 (en) * 2014-10-20 2021-01-14 Molecular Assemblies, Inc. Modified template-independent enzymes for polydeoxynucleotide synthesis
CN109477080A (zh) * 2016-06-14 2019-03-15 Dna斯克瑞普特公司 polX家族的DNA聚合酶的变体
US20200263152A1 (en) * 2017-05-22 2020-08-20 The Charles Stark Draper Laboratory, Inc. Modified template-independent dna polymerase
CN112105725A (zh) * 2018-01-08 2020-12-18 Dna斯克瑞普特公司 末端脱氧核苷酸转移酶的变体及其用途
CN112746063A (zh) * 2019-10-29 2021-05-04 中国科学院天津工业生物技术研究所 核苷转移酶的新功能及应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 21 April 2019 (2019-04-21), "DNA nucleotidylexotransferase [Podarcis muralis] ", XP093330873, Database accession no. XP_028584377 *
LU XIAOYUN, LI JINLONG, LI CONGYU, LOU QIANQIAN, PENG KAI, CAI BIJUN, LIU YING, YAO YONGHONG, LU LINA, TIAN ZHENYANG, MA HONGWU, W: "Enzymatic DNA Synthesis by Engineering Terminal Deoxynucleotidyl Transferase", ACS CATALYSIS, AMERICAN CHEMICAL SOCIETY, US, vol. 12, no. 5, 4 March 2022 (2022-03-04), US , pages 2988 - 2997, XP093330884, ISSN: 2155-5435, DOI: 10.1021/acscatal.1c04879 *

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