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A method for targeted cleavage of arbitrary nucleic acids

An endonuclease and nucleic acid technology, applied in the field of molecular biology, can solve the problems of severe non-specific cutting, cytotoxicity, etc., and achieve strong versatility and good innovative effects

Active Publication Date: 2017-04-19
NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, the non-specific cleavage of ZFNs is severe, and often miscuts lead to cytotoxicity
These all prove that the method of frequently changing the functional domain with sequence recognition activity in the endonuclease to realize the recognition of multiple sequences has great limitations

Method used

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  • A method for targeted cleavage of arbitrary nucleic acids
  • A method for targeted cleavage of arbitrary nucleic acids
  • A method for targeted cleavage of arbitrary nucleic acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1

[0049] Gene A encoding the recognition domain was amplified according to the PCR method described below. The pET24a(+)-FEN1 plasmid template (SEQ ID NO.9), 0.025U / μL Primestar enzyme, 5mM Mg 2+ , 0.2 μM oligonucleotide primers (5'-TATACATATGGGTGCGGATATTGGTGA-3', which is SEQ ID No.10 and 5'-TATAGAATTCGAACCACCTCTCAAGCGT-3', which is SEQ ID No.11), and 0.2 mM dNTPs were added to the reaction solution respectively. Using a thermal cycler, the reaction solution was heat-treated at 94°C for 3 minutes, and then cycled 30 times with the program of "94°C for 30s, 60°C for 30s, and 72°C for 100s". The PCR amplification product was purified using a PCR purification kit. Attach instructions for purification and elute with 30 μL ultrapure water. After agarose electrophoresis analysis, the results were as follows: Figure 8 As shown, the lane M is the λ-EcoT14 Idigest DNA Marker, and the lane 1 is the amplified band of the PCR product. Compared with the Marker, the b...

Embodiment 2

[0050] Embodiment 2

[0051] Utilize Nde I and EcoR I enzyme-linked enzyme reaction, insert the amplified product A in Example 1 into the plasmid pET28a(+)-His-3N (i.e., SEQ ID NO.12, wherein the 1069th to 1716bp are encoding connecting peptides and the sequence of the cleavage functional domain) (whole gene synthesis of Shanghai Jierui Bioengineering Co., Ltd.) constitutes the recombinant plasmid pET28a(+)-His-A3N, and the structure of the recombinant plasmid is as follows Figure 9 shown. Transformed into the host strain ArcticExpress for preservation (purchased from China Microbial Strain Resource Bank). The strains were inoculated in 5 mL LB (containing 30 μg / mL kanamycin) liquid medium, shaken at 200 r / min at 37 °C overnight, and the plasmid was extracted by conventional methods. The extracted plasmid was treated with restriction endonucleases Xba I and Xho I, and the correctness of the plasmid was verified by agarose gel electrophoresis. The results are shown in Fig...

Embodiment 3

[0052] Embodiment 3

[0053] Using the Escherichia coli recombinant protein expression system, express the recombinant structure recognition endonuclease according to the following method. First, the recombinant plasmid prepared by the above method is transformed into the host strain ArcticExpress by a common method. The resulting transformant was inoculated into 5 mL of LB medium containing kanamycin, and incubated at 37° C. in a shaking culture until OD600 reached 0.6. The resulting culture was inoculated into 100 mL of LB medium containing kanamycin at 1%, and further incubated at 37°C with shaking until the OD600 reached 0.6. Next, isopropyl-β-thiogalactopyranoside was added to the culture at a final concentration of 0.1 mM, thereby inducing expression of the desired protein, and induced by shaking culture at 25° C. for 16 hours. Collect the cells induced to express, sonicate the bacteria, and centrifuge. Since the recombinant protein contains a histidine tag, the super...

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Abstract

The invention relates to a method for implementing targeted cleavage on arbitrary nucleic acid; a structural identification endonuclease is constructed and expressed for establishing the method. The method comprises the concrete steps: selecting a cleavage position of a target nucleic acid, and designing a nucleic acid probe complementary with target nucleic acid; adding into a reaction system, making the probe hybridized with a substrate, and forming a secondary structure which can be identified by the structural identification endonuclease; and adding the enzyme, and carrying out a cleavage reaction. The nucleic acid structure which can be identified includes a nucleic acid intrusive structure and the like; an endonuclease identification functional domain is composed of a peptide fragment which can identify a nucleic acid secondary-structure enzyme and can be selected from E.coliMuts and the like; the cleavage functional domain is selected from a cleavage function of an IIS type endonuclease; a connection fragment between the identification functional domain and the cleavage functional domain of the endonuclease is mainly composed of serine and glycine; and the method breaks through the limit of a sequence of a nucleic acid substrate, so that the method can realize effective targeted cleavage of nucleic acids with different sequences.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a method for targeted cutting of any nucleic acid. Background technique [0002] In molecular biology, restriction endonucleases are often used for targeted cleavage of nucleic acids. Restriction endonucleases are enzymes that can catalyze nucleic acid fragmentation and only act on certain positions in a certain base sequence. Restriction endonucleases are mainly divided into three categories, among which type II restriction endonucleases can recognize a specific nucleotide sequence and cut the double strand at a fixed position in or outside the sequence to produce sticky or blunt ends. It is a commonly used tool enzyme. Although more than 3,700 restriction endonucleases have been developed, they can only recognize 300 different nucleic acid sequences, which is difficult to meet the needs of scientific research and application. Therefore, several methods to expand the ran...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N9/22
Inventor 周国华徐澍邹秉杰
Owner NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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