WO2024032512A1 - Transcriptase inverse, molécule d'acide nucléique et procédé de synthèse d'adnc - Google Patents

Transcriptase inverse, molécule d'acide nucléique et procédé de synthèse d'adnc Download PDF

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WO2024032512A1
WO2024032512A1 PCT/CN2023/111312 CN2023111312W WO2024032512A1 WO 2024032512 A1 WO2024032512 A1 WO 2024032512A1 CN 2023111312 W CN2023111312 W CN 2023111312W WO 2024032512 A1 WO2024032512 A1 WO 2024032512A1
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reverse transcriptase
mutations
nucleic acid
amino acid
acid molecule
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PCT/CN2023/111312
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Chinese (zh)
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吴凤依
钟淑瑶
章瑞程
杨浩
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菲鹏生物股份有限公司
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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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present disclosure relates to the technical field of reverse transcriptase mutants, and relates to a reverse transcriptase, a nucleic acid molecule and a method for synthesizing cDNA.
  • M-MLV Moloney Murine Leukemia Virus reverse transcriptase
  • RNase H activity RNase H activity
  • TdT terminal deoxynucleotidyl transferase
  • the RNA-dependent polymerase activity is used to synthesize the first-strand DNA (cDNA) that is complementary to RNA
  • the DNA-dependent polymerase activity is used to synthesize the DNA strand that is complementary to the first-strand cDNA, and finally obtains a complete cDNA double-stranded product.
  • M-MLV reverse transcriptase has been widely used in cDNA synthesis, cDNA library construction, isothermal amplification technology, etc.
  • Common reverse transcriptases include reverse transcriptase from AMV (avian myeloblastosis virus), which is a heterodimer composed of ⁇ and ⁇ subunits. The ⁇ subunit is obtained by enzymatic hydrolysis of the ⁇ subunit; similar heterodimers Source dimers also include Rous sarcoma virus (RSV) reverse transcriptase, which is composed of two subunits: p95 and p63; HIV-1 (human immunodeficiency virus) reverse transcriptase, which is composed of p66 and p52 subunits. Heterodimers.
  • RSV Rous sarcoma virus
  • wild-type M-MLV reverse transcriptase has poor thermal stability, but has better fidelity and lacks DNA endonuclease activity. , RNase H activity is low.
  • the present disclosure provides a reverse transcriptase with higher thermostability than wild-type M-MLV reverse transcriptase. Thermal stability is higher, and reverse transcriptase is more resistant to high temperatures, which is conducive to destroying the secondary structure of RNA templates under high temperature conditions, denaturing high GC templates, reducing non-specific binding of primers, improving reverse transcription efficiency, and thus obtaining more Reverse transcription products.
  • the present disclosure provides a reverse transcriptase, which has an A322V mutation compared with the amino acid sequence shown in SEQ ID NO: 1.
  • the inventor found that the reverse transcriptase with the above mutations can meet the reverse transcription needs of different RNA samples and is not affected by inhibitors in the samples.
  • amino acid sequence shown in SEQ ID NO:1 is as follows:
  • the position corresponding to position 322 of the amino acid sequence shown in SEQ ID NO: 1 refers to the position related to the three-dimensional structure of the loop structure in the wild-type M-MLV reverse transcriptase, and specifically refers to Position 322 in the amino acid sequence of the M-MLV reverse transcriptase mutant or a position in the amino acid sequence corresponding to position 322 in the amino acid sequence of the wild-type M-MLV reverse transcriptase.
  • the position at position 322 in the amino acid sequence of the M-MLV reverse transcriptase mutant corresponds to the amino acid sequence of the wild-type M-MLV reverse transcriptase can be determined by comparing or aligning the amino acid sequence of the mutant with the amino acid sequence of the wild-type The sequence is easily determined, for example using known algorithms.
  • an amino acid position as used in this disclosure refers to an amino acid position in the wild-type amino acid sequence, and includes positions in the mutant's amino acid sequence that correspond to amino acid positions in the corresponding wild-type amino acid sequence.
  • the above-mentioned reverse transcriptase corresponds to the amino acid sequence shown in SEQ ID NO: 1, and also has more than 2 mutations.
  • the above-mentioned reverse transcriptase corresponds to the amino acid shown in SEQ ID NO: 1.
  • it also has an amino acid selected from the group consisting of H8, P51, H204, N249, M289, T306, F309, One or more mutations of D524, E562, K571, D583 and T664.
  • the above-mentioned reverse transcriptase has 2-13 mutations at the same time.
  • the above-mentioned reverse transcriptase has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 mutations at the same time.
  • the reverse transcriptase corresponds to the amino acid shown in SEQ ID NO: 1 and, in addition to the A322V mutation, also has a mutation selected from H8Y, P51L, H204R, N249D, M289L, T306K, T306R, F309N , one or more mutations of D524G, E562Q, K571R, D583N and T664N.
  • the above reverse transcriptase has A322V, H204R, M289L, T306K and F309N mutations (i.e. M2) at the same time.
  • the above-mentioned reverse transcriptase has A322V, H204R, M289L, T306K, F309N, D524G, E562Q and D583N mutations (i.e. M3) at the same time.
  • the active center of RNase H is a motif composed of three amino acids, Asp-Glu-Asp, and the catalytic active site includes D524, E562, D583, and D653.
  • the reverse transcriptase targeting the A322V, H204R, M289L, T306K, F309N, D524G, E562Q and D583N mutations, the reverse transcriptase has both high thermal stability and low level RNase H activity. Excessive RNase H activity may degrade the RNA template in advance and reduce the yield of reverse transcription products. By providing a reverse transcriptase that reduces or lacks RNase H activity, it is beneficial to the reverse transcription synthesis of long cDNA.
  • the synthesis of long cDNA requires reverse transcriptase to have better processivity.
  • the inventors found that by simultaneously targeting the A322V, D524G, E562Q and D583N mutations, the long-chain processivity of reverse transcriptase can be improved and the reversal of M-MLV can be improved.
  • the reverse transcription length of the transcriptase for example, amplifies to 8k.
  • the above reverse transcriptase has A322V, F309N, H204R and T306K mutations at the same time.
  • F309N, H204R and T306K mutations it is beneficial to reduce TdT activity and improve the thermal stability of reverse transcriptase.
  • Inhibiting TdT activity can obtain products that are not doped with other nucleotides at the end of cDNA and reduce the generation of non-template paired bases. It also has A322V, F309N, H204R and T306K mutations, which makes the reverse transcriptase have both high thermal stability and low TdT activity.
  • the above-mentioned reverse transcriptase has A322V, M289L, F309N, H204R, T306K, D524G, E562Q, D583N, H8Y, P51L, N249D, K571R and T664N mutations (i.e. M4) at the same time.
  • Reverse transcriptase combines higher thermal stability, low level of RNase H activity, extremely low TdT activity and high reverse transcription efficiency.
  • SEQ ID NO: 2 The mutated amino acid sequence is shown below: SEQ ID NO: 2:
  • the present disclosure provides a nucleic acid molecule encoding the above-mentioned reverse transcriptase.
  • the present disclosure also provides an expression cassette, expression vector, recombinant cell or recombinant bacterium, which contains the above-mentioned nucleic acid molecule.
  • the above-mentioned expression cassette is connected with regulatory sequences for regulating the expression of the above-mentioned nucleic acid molecules, including but not limited to promoters, enhancers, signal peptide coding sequences, screening marker genes, terminators, and histidine tags. wait.
  • the present disclosure also provides a recombinant bacterium or recombinant cell containing the above-mentioned expression cassette or vector.
  • the recombinant cells may be competent cells, for example selected from E. coli or yeast competent cells.
  • Recombinant bacteria include but are not limited to Escherichia coli, such as BL21, DH5 ⁇ , Top10, etc.
  • reverse transcriptase provides the amino acid sequence of reverse transcriptase
  • those skilled in the art can easily imagine using genetic engineering technology or other technologies (chemical synthesis) to prepare reverse transcriptase, for example, from reverse transcriptase capable of recombinant expression of any of the above. It is easy for those skilled in the art to separate and purify the reverse transcriptase from the culture product of the recombinant cell. Based on this, no matter what technology is used to prepare the reverse transcriptase of the present disclosure, it all belongs to the protection of the present disclosure. scope.
  • the present disclosure also provides a reagent or kit, which includes the above-mentioned reverse transcriptase.
  • Reagents include, but are not limited to, compositions containing reverse transcriptase. For example, it also includes preservatives, preservatives and other auxiliaries.
  • the kit is, for example, a reverse transcription kit, including reverse transcriptase, oligo(dT), RNasin, etc.
  • the present disclosure also provides a method for synthesizing cDNA, which method includes the step of using the above-mentioned reverse transcriptase to synthesize DNA complementary to the template RNA.
  • the present disclosure also provides a method for constructing a cDNA library, including:
  • the biological sample to be tested is selected from at least one of soil, feces, blood, and serum.
  • Samples from different biological sources contain a variety of inhibitors that inhibit M-MLV RT activity, such as humic acid in soil and feces, hemoglobin in blood, various blood anticoagulants such as heparin and citrate, and guanidine in serum. , thiocyanate, ethanol, formamide, EDTA and plant acidic polysaccharides, etc. Therefore, improving the inhibitor resistance of the enzyme can more effectively expand the application range of reverse transcriptase.
  • the present disclosure is directed to a mutation in which the wild-type M-MLV reverse transcriptase has the 322nd amino acid mutated to Val (a three-letter abbreviation is used here to identify the amino acid residues), which can increase the reverse transcriptase thermal stability.
  • Thermal stability experiments and heat shock stability experiments confirmed that the reverse transcriptase with the above mutations has higher thermal stability and heat shock stability.
  • the reverse transcriptase provided by the present disclosure has higher detection sensitivity and higher detection rate for reverse transcription amplification.
  • the reverse transcriptase provided by the present disclosure has higher thermal stability and heat shock stability, and has higher detection sensitivity for reverse transcription amplification. , the detection rate is high.
  • This example provides a reverse transcriptase (mutant M1), which performs A322V amino acid substitution on the basis of wild-type M-MLV to obtain M1.
  • the mutant construction method is as follows:
  • A322V amino acid substitution was performed on the basis of wild-type M-MLV.
  • the mutation method was to synthesize the wild-type sequence and then design primers for point mutation. Connected through gene synthesis and enzyme digestion.
  • the recombinant strain E.coli BL21(DE3)/pET30a-M1-MMLV was cultured at 37 degrees and 220 rpm until the OD600 was about 0.6 to 0.8, pre-cooled on ice for 30 minutes, added with a final concentration of 1mM IPTG, and harvested after induction at 28 degrees for 4 hours. bacteria.
  • the cells were resuspended in lysis buffer, disrupted by sonication, purified through nickel columns, heparin, and S cation exchange columns, and then stored in 200mM potassium phosphate, 0.05% (v/v) Triton X-100, 50% glycerol, 0.01mM EDTA, 1mM DTT. That is, M1-MMLV is obtained.
  • This example provides a reverse transcriptase mutant M2, which is based on M1-MMLV in Example 1 and undergoes H204R, M289L, T306K and F309N amino acid substitutions to obtain M2.
  • the mutant construction method is as shown in Example 1, the only difference lies in the mutation sequence, and the other steps are the same.
  • This embodiment provides a reverse transcriptase mutant M3, which is based on the reverse transcriptase mutant M2 of Example 2 and undergoes D524G, E562Q and D583N amino acid substitutions to obtain M3.
  • the mutant construction method is as shown in Example 1, the only difference lies in the mutation sequence, and the other steps are the same.
  • This example provides a reverse transcriptase mutant GS-MMLV, which is based on the reverse transcriptase mutant M3 of Example 3 and undergoes H8Y, P51L, N249D, K571R and T664N amino acid substitutions to obtain GS-MMLV (i.e. M4).
  • the mutant construction method is as shown in Example 1, the only difference lies in the mutation sequence, and the other steps are the same.
  • Wild-type M-MLV was prepared using the gene synthesis method of Example 1.
  • the heat resistance of the reverse transcriptase in Examples 1-4 and Comparative Example 1 was evaluated respectively.
  • the reverse transcriptase in each example and comparative example was subjected to the following treatments: (1) direct activity detection, (2) activity detection after being placed in a constant temperature incubator at 37 degrees for 7 days, (3) treatment at 50 degrees for 8 hours Then perform activity detection.
  • qPCR system for thermal stability testing (the components used in the qPCR system are all from Feipeng Biotechnology, product number MD013):
  • Primer-probe mixture 2.5 ⁇ l
  • Reverse transcriptase 0.4 ⁇ l (200U/ ⁇ l);
  • 02F (SEQ ID NO: 4): ATGGCAGTATTCATTCA.
  • 02R (SEQ ID NO: 6): ATTATGTCTATTATTCTTT.
  • Q-PCR program 95°C for 3 minutes, (95°C for 15 seconds, 60°C for 15 seconds, 72°C for 15 seconds to read the fluorescence signal) ⁇ 40 cycles.
  • NoCt in the table means: no amplification.
  • Sensitivity detection qPCR system (the components used in the qPCR system are all from Feipeng Biotech, product number MD013):
  • Primer-probe mixture 2.5 ⁇ l
  • Reverse transcriptase 0.4 ⁇ l (200U/ ⁇ l);
  • Q-PCR program 95°C for 3 minutes, (95°C for 15 seconds, 60°C for 15 seconds, 72°C for 15 seconds to read the fluorescence signal) ⁇ 40 cycles.
  • NoCt in the table means: no amplification.
  • the present disclosure provides a reverse transcriptase, a nucleic acid molecule, and a method for synthesizing cDNA.
  • This reverse transcriptase increases thermal stability, which is beneficial to destroying the secondary structure of RNA templates under high temperature conditions, denaturing high GC templates, reducing non-specific binding of primers, improving reverse transcription efficiency, and thus obtaining more reverse transcription products. , has broad application prospects and high market value.

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Abstract

La présente invention concerne le domaine technique des mutants de transcriptase inverse, et plus particulièrement une transcriptase inverse, une molécule d'acide nucléique et un procédé de synthèse d'un ADNc. En introduisant des mutations dans la séquence d'acides aminés de la transcriptase inverse M-MLV de type sauvage, la présente invention améliore considérablement la stabilité thermique de la transcriptase inverse. La stabilité thermique et les dosages de stabilité au choc thermique ont démontré qu'une transcriptase inverse portant les mutations présente une stabilité thermique et une stabilité au choc thermique plus élevées. Par rapport à la transcriptase inverse de type sauvage, la transcriptase inverse selon l'invention présente une sensibilité plus élevée et des taux de détection élevés dans des détections d'amplification de transcription inverse.
PCT/CN2023/111312 2022-08-11 2023-08-04 Transcriptase inverse, molécule d'acide nucléique et procédé de synthèse d'adnc WO2024032512A1 (fr)

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CN202210963198.8 2022-08-11
CN202210963198.8A CN117586983A (zh) 2022-08-11 2022-08-11 一种逆转录酶、核酸分子及合成cDNA的方法

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1430670A (zh) * 2000-05-26 2003-07-16 茵维特罗根公司 热稳定逆转录酶及其用途
CN106164261A (zh) * 2014-01-22 2016-11-23 生命技术公司 适用于高温核酸合成的新颖逆转录酶
CN107058258A (zh) * 2008-04-10 2017-08-18 赛默飞世尔科技波罗的海Uvb公司 一种逆转录酶和编码其的多核苷酸
CN111647576A (zh) * 2020-06-24 2020-09-11 南京诺唯赞生物科技股份有限公司 一种热稳定性逆转录酶突变体及其应用
WO2021119320A2 (fr) * 2019-12-11 2021-06-17 10X Genomics, Inc. Variants de transcriptase inverse

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1430670A (zh) * 2000-05-26 2003-07-16 茵维特罗根公司 热稳定逆转录酶及其用途
CN107058258A (zh) * 2008-04-10 2017-08-18 赛默飞世尔科技波罗的海Uvb公司 一种逆转录酶和编码其的多核苷酸
CN106164261A (zh) * 2014-01-22 2016-11-23 生命技术公司 适用于高温核酸合成的新颖逆转录酶
WO2021119320A2 (fr) * 2019-12-11 2021-06-17 10X Genomics, Inc. Variants de transcriptase inverse
CN111647576A (zh) * 2020-06-24 2020-09-11 南京诺唯赞生物科技股份有限公司 一种热稳定性逆转录酶突变体及其应用

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