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WO2025137872A1 - Mutant de désoxyribonucléoside transférase terminale, procédé de préparation s'y rapportant et utilisation associée - Google Patents

Mutant de désoxyribonucléoside transférase terminale, procédé de préparation s'y rapportant et utilisation associée Download PDF

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Publication number
WO2025137872A1
WO2025137872A1 PCT/CN2023/142043 CN2023142043W WO2025137872A1 WO 2025137872 A1 WO2025137872 A1 WO 2025137872A1 CN 2023142043 W CN2023142043 W CN 2023142043W WO 2025137872 A1 WO2025137872 A1 WO 2025137872A1
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WO
WIPO (PCT)
Prior art keywords
deoxyribonucleoside
terminal
combination
nucleic acid
transferase
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Pending
Application number
PCT/CN2023/142043
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English (en)
Chinese (zh)
Inventor
杨卫康
于爱淼
谢庆庆
郑越
沈玥
徐讯
董宇亮
章文蔚
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bgi Changzhou
BGI Shenzhen Co Ltd
Original Assignee
Bgi Changzhou
BGI Shenzhen Co Ltd
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Filing date
Publication date
Application filed by Bgi Changzhou, BGI Shenzhen Co Ltd filed Critical Bgi Changzhou
Priority to PCT/CN2023/142043 priority Critical patent/WO2025137872A1/fr
Priority to PCT/CN2024/140779 priority patent/WO2025140014A1/fr
Publication of WO2025137872A1 publication Critical patent/WO2025137872A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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 belongs to the field of genetic engineering technology and relates to a terminal deoxyribonucleoside transferase mutant and a preparation method and application thereof.
  • Phosphoramidite synthesis is the mainstream method for synthesizing short-chain DNA, which can reliably provide short DNA chains of ⁇ 200 nucleotides.
  • Phosphoramidite synthesis 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 long single cycle time (6-8 minutes), high consumption of chemical reagents, and high cost. In addition, a large amount of toxic and flammable organic reagents are used in the reaction process, which causes great pollution.
  • the synthesis of nucleotide sequences >200bp by phosphoramidite synthesis is still a heavy burden. For this reason, researchers have tried to develop alternative technologies such as Gibbs assembly to produce longer DNA chains.
  • 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
  • TdT extends oligonucleotides in a promiscuous manner in the 5' to 3' direction of the starting chain. Since any of the four nucleotides will participate in each step of the reaction, it will eventually lead to the simultaneous formation of oligonucleotide sequences of different lengths and codes.
  • an effective solution is to control the incorporation of nucleotides through a "reversible termination" mechanism: in the pentose sugar of the nucleotide, the incorporation of nucleotides is controlled by a "reversible termination” mechanism.
  • a reversibly removable protecting group "Protecting Group, PG” is added to the 3rd position of the nucleotide to ensure that each reaction step is terminated after extending one nucleotide, and in the subsequent reaction, PG can be removed and restored to a hydroxyl group to extend the next required nucleotide ( Figure 1).
  • This DNA de novo synthesis strategy catalyzed by TdT only requires two steps of coupling and deprotection to complete a single base addition cycle. Compared with the phosphoramidite synthesis method, it greatly improves the possible limit of single-cycle efficiency, and the entire reaction is completed in the aqueous phase, with the characteristics of mild reaction conditions and greener.
  • CN114921436A discloses a terminal deoxynucleotidyl transferase mutant with improved thermal stability, by mutating the sites of the amino acid sequence of the wild-type terminal deoxynucleotidyl transferase as shown in SEQ ID NO.1 to obtain a mutant with six mutations: N135P, S138H, Q229D, K232D, L234R, and V366M.
  • the present application provides a terminal deoxyribonucleoside 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 deoxyribonucleoside transferase.
  • the activities of the GeTdT mutants shown in the figure for the four nucleotides with azidomethyl modification at the 3' end are better than those of the wild type, among which the conversion rates of the mutants E459R, E459R/R460Q, E459R/R460K, E459R/R460E, E459R/R460H, E459R/R460N and E459R/R460D are greatly improved compared with the wild type.
  • the oligonucleotide substrate 5'-ROX-Oligo(dT) 98 was extended by one base under the action of terminal deoxyribonucleoside transferase to obtain the product 5'-ROX-Oligo(dT) 99 -3'-O-Azidomethyl.
  • the reaction system for determining the conversion rate of the GeTdT wild type and its mutants is shown in Table 3. After the reaction system is mixed, the reaction is reacted at 37°C for 10 minutes. After the reaction is completed, the reaction is terminated by heating at 95°C for 10 minutes.
  • Oligo(dT) 18 an oligonucleotide substrate, was extended by one base under the action of terminal deoxyribonucleoside transferase to obtain the product Oligo(dT) 19 -3'-O-PG.
  • the reaction system for the terminal transfer activity test of the wild-type GeTdT enzyme and its mutants is shown in Table 5. After the reaction system is mixed, it is reacted at 37°C for 10 minutes. After the reaction is completed, it is heated at 95°C for 10 minutes to terminate the reaction.
  • the reaction products of GeTdT wild type and its mutants were semi-quantitatively analyzed by urea polyacrylamide gel electrophoresis (20% denaturing gel). The results are shown in FIG5 .
  • NC represents Oligo(dT) 18 ; Lanes 1-3 represent: 1, WT; 2, E459R; 3, E459R/R460E.
  • the 5 ⁇ reaction buffer used in this application is formulated as: 0.5M Na-Cacodylate, 5mM CoCl 2 , pH 7.2.
  • the present application obtains advantageous TdT mutants by molecularly modifying natural TdT, thereby greatly improving the catalytic efficiency of modified nucleic acid monomers and achieving efficient single-base addition efficiency.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un mutant de désoxyribonucléoside transférase terminale, un procédé de préparation s'y rapportant et une utilisation associée. Une désoxyribonucléoside transférase terminale de type sauvage est soumise à une modification moléculaire, pour améliorer l'activité de polymérisation de monomère d'acide nucléique modifié et l'efficacité de catalyse de monomère d'acide nucléique modifié, ainsi que pour réaliser une efficacité d'addition à base unique efficace. De multiples substrats de dNTP modifiés bloquant 3'-O peuvent être ajoutés à une extrémité 3'-OH d'une chaîne unique oligonucléotidique sans matrice, de telle sorte qu'une nouvelle enzyme outil efficace peut être fournie pour le procédé enzymatique à partir de la synthèse de novo d'acide nucléique.
PCT/CN2023/142043 2023-12-26 2023-12-26 Mutant de désoxyribonucléoside transférase terminale, procédé de préparation s'y rapportant et utilisation associée Pending WO2025137872A1 (fr)

Priority Applications (2)

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

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
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

Publications (1)

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WO2025137872A1 true WO2025137872A1 (fr) 2025-07-03

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PCT/CN2023/142043 Pending WO2025137872A1 (fr) 2023-12-26 2023-12-26 Mutant de désoxyribonucléoside transférase terminale, procédé de préparation s'y rapportant et utilisation associée
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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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 (3)

* 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
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
WO2023143123A1 (fr) * 2022-01-28 2023-08-03 中国科学院天津工业生物技术研究所 Variant de transférase terminale pour la synthèse contrôlable d'adn simple brin et son utilisation

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11390858B2 (en) * 2014-10-20 2022-07-19 Molecular Assemblies, Inc. Modified template-independent enzymes for polydeoxynucleotide synthesis
WO2018217689A1 (fr) * 2017-05-22 2018-11-29 The Charles Stark Draper Laboratory, Inc. Adn polymérase indépendante d'une molécule gabarit modifiée
JP2021510074A (ja) * 2018-01-08 2021-04-15 ディーエヌエー スクリプト ターミナルデオキシヌクレオチジルトランスフェラーゼの改変体およびその使用

Patent Citations (3)

* 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
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
WO2023143123A1 (fr) * 2022-01-28 2023-08-03 中国科学院天津工业生物技术研究所 Variant de transférase terminale pour la synthèse contrôlable d'adn simple brin et son utilisation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Wanfang Dissertations of China", 27 May 2021, article TANG MENGTONG: "The Study on the Regulation of TdT Enzymatic DNA Polymerization", XP093329885, DOI: 10.27356/d.cnki.gtjdu.2021.002500 *
DATABASE PROTEIN 26 February 2023 (2023-02-26), XP093329940, Database accession no. XP_053243387.1 *
GUO MIAO, WANG YINA, TANG YUYUE, CHEN ZIJING, HOU JINFENG, DAI JINGLI, WANG YUDONG, WANG LIANGYAN, XU HONG, TIAN BING, HUA YUEJIN,: "Mechanism of genome instability mediated by human DNA polymerase mu misincorporation", NATURE COMMUNICATIONS, NATURE PUBLISHING GROUP, UK, vol. 12, no. 1, UK, XP093329943, ISSN: 2041-1723, DOI: 10.1038/s41467-021-24096-7 *
TROSHCHYNSKY, A. ET AL.: "Functional Analyses of Polymorphic Variants of Human Terminal Deoxynucleotidyl Transferase", GENES AND IMMUNITY, vol. 16, 4 June 2015 (2015-06-04), XP036971149, DOI: 10.1038/gene.2015.19 *

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