WO2013102290A1 - Procédé pour reconnaître spécifiquement de l'adn contenant 5-cytosine méthylée - Google Patents

Procédé pour reconnaître spécifiquement de l'adn contenant 5-cytosine méthylée Download PDF

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WO2013102290A1
WO2013102290A1 PCT/CN2012/001718 CN2012001718W WO2013102290A1 WO 2013102290 A1 WO2013102290 A1 WO 2013102290A1 CN 2012001718 W CN2012001718 W CN 2012001718W WO 2013102290 A1 WO2013102290 A1 WO 2013102290A1
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dna
protein
dhax3
tale
cytosine
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PCT/CN2012/001718
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施一公
颜宁
邓东
闫创业
潘孝敬
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清华大学
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of biotechnology, and more particularly to a method for specifically recognizing DNA containing 5-methylcytosine. Background technique
  • TALE Transcription Activator Like Effectors
  • TALE Transcription Activator Like Effectors
  • TALE family proteins generally consist of three major functional domains, the N-terminal domain and
  • TALE is involved in secretion transport; the C-terminus has a transcriptional activation domain and a nuclear signal peptide fragment; the region located in the middle of TALE is a DNA-binding domain, but its DNA-binding domain is different from other known DNA-binding domains. It consists of a series of repeating units. In most cases, each repeat unit consists of 34 amino acids, and individual repeat units consist of 33 0 or 35 amino acid residues. Among the 34 amino acids, except for the amino acid changes at positions 12 and 13, the other amino acids are highly conserved. These two non-conservative amino acids are named RVD (repeat variable diresidue, repeated variable double residues). J. Boch et al. and MJ Moscou et al. (see J.
  • TALE Transcription activator-like effectors
  • the invention relates to a method for detecting cytosine thiolation in DNA, comprising the use of TALE protein and a derivative thereof to specifically recognize 5-mercapto-cytosine in DNA.
  • two different TALE proteins are used to specifically recognize cytosine and 5-mercapto-cytosine in the target sequence.
  • the method is for detecting guanidation of a CpG island.
  • the invention relates to the use of a TALE protein and a protein thereof for the specific recognition of 5-nonylated cytosine in DNA.
  • the invention relates to the use of a TALE protein and a derivative thereof for the preparation of a reagent for the specific recognition of 5-mercaptocytosine in DNA.
  • the invention relates to the use of a TALE protein and a derivative thereof for the preparation of a medicament for the diagnosis or treatment of cancer.
  • the diagnosis or treatment is carried out by specifically recognizing 5-mercapto-cytosine in the DNA.
  • the invention further relates to TALE proteins and derived proteins thereof for the specific recognition of 5- Amidoxime-modified DNA.
  • the invention also relates to TALE proteins and derived proteins thereof for use in the diagnosis or treatment of cancer.
  • the TALE protein can be a natural TALE protein and a TALE-derived protein that retains or enhances the specific recognition of 5-methylcytosine in DNA by genetic modification, modification, and assembly.
  • the TALE-derived protein further comprises a recombinant protein having a TALE protein DNA binding domain.
  • Figure 1 is a schematic representation of the high-resolution crystal structure (1.85 angstrom) of the DNA binding domain of dHax3 (dHax3 truncated, labeled dHax3-A) and double-stranded DNA.
  • 1-10 in the left panel shows each repeat unit of the DNA binding domain of dHax3, which recognizes the corresponding DNA sequence on the right side.
  • Each repeating unit consists of two o helices, and the two helices are and 1>, respectively.
  • the structure has been uploaded to the PDB database with the code: 3V6T.
  • dHax3 (designed Hax3 ) refers to the modified TALE protein Hax3.
  • Figure 2 shows the interaction between dHax3 and DNA bases.
  • A the side chain of RVD in dHax3 is pointed, the first amino acid in RVD does not extend into the DNA major groove, and the second amino acid extends the amino acid side chain to the DNA major groove;
  • B the first amino acid in RVD passes Hydrogen bond stabilizes the loop region conformation.
  • the first amino acid of the DNA binding domain repeat unit is asparagine (N) or histidine (H)
  • they are the eighth amino acid backbone of the repeat sequence.
  • the carbonyl oxygen atom forms a hydrogen bond interaction, which acts to stabilize the loop conformation of the entire RVD;
  • C the second amino acid in RVD directly interacts with the DNA base, when the amino acid residue is aspartic acid (D)
  • D When the carboxyl oxygen of aspartic acid directly forms a hydrogen bond with the amino group of cytosine in DNA through hydrogen bonding;
  • S serine
  • S the hydroxyl group in serine forms direct hydrogen with N7 in adenine.
  • Figure 3 is a comparison of the structure of thymine (left) and 5-mercapto-cytosine (right). It is clear from the comparison in the figure that the only difference between thymine (left) and 5-mercapto-cytosine (right) is the amino group at the six position and the carbonyl oxygen atom. Both the amino group and the carbonyl oxygen atom may interact with the amino acid residues of the protein by van der Waals forces.
  • Figure 4 shows that biochemical experiments and crystal structure analysis revealed that TALE protein recognizes 5-mercapto-cytosine through NG.
  • dHax3-recognized DNA sequence containing 5-mercapto-cytosine (5mC) (this sequence is called dHax3-5mC, contains three 5mC, only shows the base recognized by RVD of dHax3, the specific sequence is shown in the examples) And the corresponding RVD in the dHax3 protein; b.
  • EMSA detects the dHax3 pair of DNA sequences without 5mC (called dHax3 box, which is identical to the dHax3-5mC sequence except for 5mC) and the dHax3 pair of 5mC DNA sequences (dHax3) -5mC) binding capacity, about 4 nM nucleic acid probe was added to each lane; and gradient concentrations of dHax3 protein were added to the lanes 0 to 10, respectively, concentration 0, 8nM, 16nM, 31.5nM, 62.5nM , 125nM, 250nM, 500nM, ⁇ ⁇ ,
  • Figure 5 is an electropherogram showing the purification results of the dHax3 full-length protein. Lane marking
  • Ming 1. Whole bacteria breaking liquid; 2. Whole bacteria crushing and centrifugation; 3. Whole bacteria crushing centrifugal supernatant; 4. Nickel column culture waste liquid; 5. Nickel column cleaning liquid; 6. Nickel column elution recovery Liquid; 7. Nickel column; 8. Molecular weight marker.
  • Figure 6 is an electropherogram showing the purification results of the dHax3 truncated body protein (dHax3-A).
  • FIG. 7 shows that DNA binding experiments demonstrate that NG can specifically recognize thiolated cytosine.
  • 6T-6C indicates that 6 thymines (T) in dHax3-box were replaced with 6 cytosines (C);
  • 6T-6mC means 6 thymines (T) in dHax3-box with 6 methyl groups Cytosine (5mC) substitution;
  • 5C-5mC means replacing 5 cytosines (C) in dHax3-box with 5 methylated cytosines (5mC);
  • 5C-5mC means 5 of dHax3-box Cytosine (C) was replaced with 5 methylated cytosines (5mC);
  • 5C-5T was substituted for 5 cytosines (C) in dHax3-box with 5 thymine (5T) 0; 5C-5A Five cytosines
  • dHax3 has a DNA sequence containing six thiolation modifications (6T-6mC) Similar binding ability to the control experiment (dHax3-box).
  • 6T-6mC six thiolation modifications
  • c an RVD in dHax3, NG, cannot bind to cytosine (C) without thiolation.
  • d an RVD in dHax3 - HD - is specifically recognized for cytosine (C), and methylation modification affects the recognition of HD and cytosine.
  • Figure 8 is the DNA binding domain of dHax3-NN variant (dHax3-NN-A, that is, the RVD (NS) in the seventh repeat unit of the DNA binding domain of dHax3 is changed to NN by point mutation technique and the ninth RVD (HD) in the repeat unit is changed to NN by point mutation technique to form recognition of two thiolated CpG islands, and the specific recognition sequence thereof is shown in the examples.)
  • the crystal structure of the DNA containing two methylated CpG islands is combined. . detailed description
  • the inventors successfully analyzed the crystal structure of the complex of the DNA binding domain of the modified TALE protein Hax3 (referred to herein as dHax3 (designed Hax3)) and dsDNA.
  • dHax3 modified Hax3
  • dsDNA dsDNA binding domain of the modified TALE protein Hax3
  • NG in RVD relies on van der Waals force and 5-methyl interaction of thymine, and other thymine groups do not participate in the reaction.
  • the TALE protein may specifically recognize 5-methylcytosine in the DNA Han chain through NG because 5-mercapto-cytosine has a similar structure to thymine.
  • the inventors also successfully resolved the crystal structure of the complex of the DNA binding domain of dHax3 and the dsDNA having 5-mercaptocytosine.
  • This discovery provides a novel method for detecting and interfering with cytosine thiolation and can be used in the following ways:
  • DNA thiolation refers to the covalent bond of a methyl group at the 5' carbon position of the cytosine of the genomic CpG dinucleotide under the action of a DNA thiotransferase. Due to the close relationship between DNA thiolation and human development and tumor diseases, especially the transcriptional inactivation of tumor suppressor genes caused by thiolation of CpG islands,
  • methylation regions appear in the genome of cancer cells, and these thiolation phenomena do not occur in normal cells. Since the method of the present invention can effectively distinguish whether methylation occurs at a specific genomic locus, it can be used as a new cancer cell. testing method.
  • DNA methylation of cancer cells inhibits the expression of many tumor suppressor genes. Since the method of the present invention specifically reopens the expression of these genes in cancer cells, it can promote apoptosis of cancer cells.
  • TALE itself has the function of activating transcription. By designing the RVD on the TALE repeat sequence, it specifically binds to the upstream promoter sequence of the thiolated modified tumor suppressor gene, specifically opening up a large number of tumor suppressor genes in cancer cells. , to achieve the purpose of killing cancer cells.
  • TALE protein refers to Transcription Activator like
  • the TALE protein can be a natural TALE protein and a TALE-derived protein which retains or enhances the DNA, or DNA-RNA hybrid chain binding ability obtained by genetic modification, modification, and assembly.
  • Hax3 refers to one of the members of the TALE protein family.
  • the full name of Hax 0 is "Homolog of avrBs3 in Xanthomonas", t3 ⁇ 4 Hax3 ⁇ Aff > ⁇ 3 ⁇ 4 ⁇ "
  • One of the three homologous proteins identified by Armor aciae Xanthomonas campestris pv. Armoraciae ).
  • One of its members, its function is similar to that of other known TALE proteins such as avrBs3 (see S. Kay, J. Boch, U.
  • dHax3 refers to an artificially engineered Hax3 (designed Hax3) whose nucleotide sequence is SEQ ID NO: 1 and the amino acid sequence can be found in SEQ ID NO: 20 (with a 6XHis tag inserted therein) MM Mahfouz et al. designed dHax3 to have the ability to specifically recognize the following DNA sequences: TCCCTTTATCTCT (MM Mahfouz, L. Li, M. Shamimuzzaman, A. Wibowo, X. Fang, JK Zhu, De Novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks,
  • TALE transcription activator-like effector
  • dHax3 truncated body protein (“dHax3-A”) as used herein refers to a dHax3 truncated protein that has a N-terminal domain and a C-terminal domain removed, which is the dHax3 protein sequence.
  • dHax3-NN variant refers to a variant of dHax3 in which the RVD (NS) in the seventh repeat unit of the DNA binding domain of dHax3 is converted by a point mutation: G technology becomes NN and ninth RVD (HD) in a repeating unit becomes a point mutation technique
  • dHax3-NN-A refers to the protein sequence of the dHax3-NN variant.
  • a truncated body of 230-721, that is, a DNA binding domain is retained.
  • RVD-NG which involves dHax3 in the examples, specifically recognizes cytosine thiolation.
  • the ability to apply equally to other TALE proteins other than the dHax3 sequence of the examples is also within the scope of this patent.
  • composition of the 50 ⁇ standard PCR reaction system is shown in the following table, and the system can be amplified according to the ratio if necessary;
  • the amplified target gene fragment was directly recovered using a common DNA recovery kit. Note that if the amplified gene fragment is a point mutation, the DNA template is first removed by agarose gel electrophoresis, and then the target gene is recovered using an agarose gel DNA recovery kit.
  • the amplified fragment and vector were treated with the same restriction endonuclease to generate the same DNA cohesive ends.
  • the composition of the 50 ⁇ double digestion reaction system is shown in the following table:
  • the DNA fragment is recovered by gel electrophoresis of the lipogel gel DNA recovery kit.
  • the digested target gene fragment was ligated into the vector at room temperature for 30 to 120 min using T4 DNA ligase.
  • the connection system is shown in the following table:
  • the ligation product was transferred into DH5a competent cells according to the following method, and the positive clones were prepared for screening: 50 ⁇ 100 ⁇ 1 DH5 competent cells were added to the ligation product, placed on ice for 30 min; heat shocked at 42 °C for 90 s; placed on water for 2 min; All products were applied to ampicillin-resistant agar plates, spread with a coating bar, and cultured in an inverted 14-16 hours.
  • the plasmid was extracted using a common plasmid mini-kit, and sequencing was performed by Genewiz Biotech Co., Ltd.
  • overexpression is required.
  • Existing overexpression systems are Escherichia coli (£. / ), yeast, insect cells, and the like. Different proteins may be suitable for expression in different systems.
  • the target protein is a protein in Gram-negative bacteria, so E. coli was selected as an expression system for protein expression purification.
  • the specific purification steps are as follows: 50 ml of LB medium containing ampicillin or ampicillin/chloramphenicol antibody was added and incubated overnight at 37 °C on a shaker.
  • the induced E. coli was centrifuged at 4400 rpm for 4 min at 10 ° C, and the supernatant was discarded.
  • the wet bacteria collected by centrifugation per liter of culture medium were resuspended in 20 ml of lytic solution (25 11 1 ⁇ butyl 1 ⁇ -1 ⁇ 1 1 8.0 8.0, 500 mM NaCl).
  • elution buffer 25 mM Tris-HCl pH 8.0, 50 mM NaCl, 300 mM Imidazole
  • elution buffer 25 mM Tris-HCl pH 8.0, 50 mM NaCl, 300 mM Imidazole
  • Coomassie Brilliant Blue G-250 to check for cleanliness. If the elution is incomplete, repeat the above procedure.
  • the protein purified by the above two-step affinity chromatography was concentrated to ⁇ 10 mg/ml using an ultrafiltration concentrating tube. Finally, the protein was further purified using a molecular sieve (Superdax 200) and the protein was used.
  • the buffer used for the molecular sieve was 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM DTT.
  • the buffer in the desalting column (Hiprep 26/10) dHax3 (231-720) protein was replaced with 25 mM MES pH 6.0, 50 mM NaCl, 5 mM MgCl 2 , 10 mM DTT.
  • the dHax3 (designed Hax3) gene is obtained by whole gene synthesis and the sequence is as follows (SEQ ID NO:
  • the synthesized gene was directly ligated into the pET300 (invitrogen) plasmid.
  • the expressed full-length protein has six histidine tags at the N-terminus and is used for affinity purification of the nickel column by protein purification.
  • the full-length protein sequence is as follows (SEQ ID NO: 2):
  • dHax3 truncation a truncated body protein (dHax3 truncation, labeled dHax3-A) containing the protein sequence 230-721) to obtain a more stable protein.
  • the dHax3 truncation was cloned into the pET21 (Novagen) expression vector.
  • the expressed dHax3 truncated protein sequence is as follows, wherein the C-terminus contains a His 6 tag for affinity purification by nickel column for protein purification (SEQ ID
  • the inventors also constructed and expressed the dHax3-NN-A protein for use with CpG islands.
  • Table 2 shows the RVD of the TALE repeat unit involved in the experiment and its identified DNA:
  • the synthesized single-stranded DNA was dissolved to 1 mM, the two single-stranded DNAs were mixed in an equimolar ratio, and the bath was heated at 85 ° C for more than 3 min, and slowly cooled to 22 ° C, which was not less than 3 hours.
  • lyophilization and cryopreservation can be performed.
  • the purified dHax3 truncated body protein (231-721 in the full-length sequence) was adjusted to a protein concentration of 6 to 7 mg/ml, and the double-stranded DNA after annealing at a molar ratio of 1.5:1 was added and incubated at 4 °C. 30 min.
  • the conditions for protein crystallization were screened from the above Kit, and the crystallization conditions were optimized by adjusting the concentration of the precipitant, the species, the concentration and type of the salt ions, and the concentration and type of the buffer.
  • the crystal was optimized using the Addtive Screen and the Detergent Screen Kit. At the same time, the crystal is dehydrated, annealed, etc. to improve the diffraction quality of the crystal.
  • Crystallization mother liquor 8-10% PEG3350 (w/v), 12% ethanol, 0.1 M MES pH 6.0. Climbing data collection and processing
  • Width allowed 7.3 6.5 Generously allowed 0.0 0.0
  • dHax3-A and double-stranded DNA dsDNA
  • This structure clearly demonstrates that dHax3 exhibits a right-handed helical structure that wraps dsDNA in the middle of the entire complex.
  • the protein is entangled outside the DNA and embedded in the large 5 groove of DNA (see Figure 1).
  • the structure shows that the 12th amino acid (histidine/asparagine) located in each repeat does not directly interact with DNA, but instead they will be the 8th amino acid (alanine) of the repeat sequence in which they are located.
  • the main chain oxygen atom forms a hydrogen bond, which acts to fix the ring in which the entire RVD is located.
  • thymine (T) and 5-methylcytosine (5mC) indicate that 5-mercapto-cytosine has a sulfhydryl group in the fifth position, and this thiol group is the only group recognized by NG. Therefore, NG may recognize 5mC. Accordingly, the inventors designed the DNA sequence dHax-5mC (Fig. 4a)
  • the inventor designed the DNA sequence dHax3-CpG.
  • Table 4 Crystal structure of the complex of dHax3-A and dHax3-5mC and statistical data of data collection and structural correction of the crystal structure of the complex of dHax3-NN-A and :0 dHax3-CpG Data DNA (dHax3-5mC) binds DNA (dHax3-CpG) to bind dHax3-A's dHax3-NN-A
  • the inventors analyzed the complex structure of dHax3 protein with three 5mC DNA with a resolution of 1.85 angstroms.
  • the high-resolution structure clearly reveals the molecular mechanism by which the dHax3 protein recognizes mC (Fig. 4c).
  • Figure 8 shows a schematic diagram of the DNA binding domain of the dHax3-NN variant and the crystal structure of the DNA containing two thiolated CpG islands, which confirmed that the dHax3-NN-A binding contains two thiolated CpG island DNAs.
  • DNA thiolation occurs only on C in CpG islands.
  • the applicant analyzed the crystal structure of TALE and the DNA sequence containing two CpG islands, further demonstrating that TALE has a specific recognition ability for thiolated DNA. This is very important for the expansion of TALE applications.
  • Example 4 Gel retardation assay demonstrates the ability of dHax3 to bind to DNA duplexes with 5-mercaptocytosine (5mC)
  • the gel retardation assay is a special gel electrophoresis technique that studies the interaction of DNA/RNA with proteins in vitro.
  • the basic principle is: In gel electrophoresis, due to the action of the electric field, the nucleic acid fragment of a small molecule moves faster toward the anode than the nucleic acid fragment to which the protein is bound. Therefore, a short nucleic acid fragment can be labeled, mixed with a protein, and the mixture can be subjected to gel electrophoresis. If the target DNA binds to a specific protein, the speed of movement is retarded, and autoradiography of the gel can be found. Nucleic acid binding protein. At the same time, by statistically comparing the amount of DNA bound to the protein and the amount of DNA of the unbound protein, a more accurate fit calculation can be made to the binding affinity of the protein to the nucleic acid.
  • 6T 6C 5 -CCACATATGTCATACGTGTCCCCCCACCCCCCTCCAGCTCGAG
  • T4 polynucleotide kinase (lOU/ ⁇ ) 1 ⁇ After setting up the reaction system according to the above table, gently mix and incubate at 37 °C for 30 min; use G25 pre-installed desalting column to remove excess [ ⁇ ] - 32 ⁇ ]- ⁇ , adding an excess of unlabeled complementary strands, annealing to generate double-stranded DNA or DNA-RNA hybrid double strands.
  • reaction components were added to the reaction system in the above ratio, and mixed for 4 min at 4 ° C; the reacted sample was run 6 % non-denaturing gel;
  • Image data was read with a Typhoon 9400 varible scanner.
  • FIG. 7 shows an RVD in dHax3 - NG - unable to bind to a cytosine without thiolation modification; and an RVD - HD in dHax3 - is specifically recognized for cytosine (C), and Methylation of cytosine affects the recognition of HD and cytosine.

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Abstract

L'invention concerne un procédé permettant de reconnaître spécifiquement l'ADN contenant 5-cytosine méthylée. Le procédé consiste à reconnaitre 5 - cytosine méthylée dans l'ADN en utilisant la protéine TALE.
PCT/CN2012/001718 2012-01-04 2012-12-21 Procédé pour reconnaître spécifiquement de l'adn contenant 5-cytosine méthylée WO2013102290A1 (fr)

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WO2015052335A1 (fr) * 2013-10-11 2015-04-16 Cellectis Procédés et kits de détection de séquences d'acide nucléique d'intérêt à l'aide d'un domaine protéique de liaison à l'adn
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CN109384833A (zh) * 2017-08-04 2019-02-26 北京大学 特异性识别甲基化修饰dna碱基的tale rvd及其应用
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CN105154558B (zh) * 2015-09-22 2018-10-09 武汉大学 一种检测dna中甲基化胞嘧啶的方法

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015052335A1 (fr) * 2013-10-11 2015-04-16 Cellectis Procédés et kits de détection de séquences d'acide nucléique d'intérêt à l'aide d'un domaine protéique de liaison à l'adn
CN104498594A (zh) * 2014-12-04 2015-04-08 李云英 TALEs双识别检测方法及其应用
WO2019024081A1 (fr) * 2017-08-04 2019-02-07 北京大学 Rvd de tale reconnaissant spécifiquement une base d'adn modifiée par méthylation et application correspondante
CN109384833A (zh) * 2017-08-04 2019-02-26 北京大学 特异性识别甲基化修饰dna碱基的tale rvd及其应用
CN111278848A (zh) * 2017-08-04 2020-06-12 北京大学 特异性识别甲基化修饰dna碱基的tale rvd及其应用
US11897920B2 (en) 2017-08-04 2024-02-13 Peking University Tale RVD specifically recognizing DNA base modified by methylation and application thereof
US11624077B2 (en) 2017-08-08 2023-04-11 Peking University Gene knockout method

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