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Artificial endo enzyme containing peptide nucleic acid recognition element, and its preparing method and use

A technology for recognizing elements and endonucleases, applied in the fields of molecular biology and enzymology, can solve problems such as the lack of DNA substrate specificity or selective cleavage

Inactive Publication Date: 2005-02-16
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, people such as Shen Hebai disclosed the phosphodiester bond in the Ce ion hydrolyzable oligonucleotide DNA in Chinese Science Volume 31, No. 2 (April 2001), but all did not realize the DNA substrate (target sequence) Specific or selective cleavage of

Method used

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  • Artificial endo enzyme containing peptide nucleic acid recognition element, and its preparing method and use
  • Artificial endo enzyme containing peptide nucleic acid recognition element, and its preparing method and use
  • Artificial endo enzyme containing peptide nucleic acid recognition element, and its preparing method and use

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preparation example Construction

[0041]Methods for preparing peptide nucleic acids are well known in the art and are described in numerous documents, for example Oliver Seitz, "Solid Phase Synthesis Of Protected Peptide Nucleic Acid" Tetrahedron Letters 40, 4161-4164, 1999 and Birgitte Hyrup et al., "Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Application", Bioorganic & Medicinal Chemistry, Vol.4, No.1, pp.5-23, 1996.

[0042] In the present invention, the length of the identification element is not particularly limited. Depending on the use of the artificial endonuclease, the length of the recognition element can be at least 4 amino acid structural units, preferably 6-200 structural units, more preferably 8-100 structural units, and most preferably 10- 50 structural units. A particularly preferred recognition element is peptide nucleic acid (abbreviated "PNA"). Due to the development of PCR synthesis technology, DNA synthesis technology and many isolation technologies, those skilled in...

Embodiment 1

[0067] Synthesis of Artificial Endonuclease

[0068] The recognition element is a peptide nucleic acid composed of 10 amino acid structural units, and its sequence is H-GTACAGTGAA-Lys-NH 2 (from N'→C', N' corresponds to the 5' end of the nucleic acid, and C' corresponds to the 3' end of the nucleic acid), purchased from Chengdu Paide Biotechnology Co., Ltd. ) molecule of PNA, forming the PNA-lys-NH 2 .

[0069] With the sequenced PNA, press figure 1 The route shown is for the synthesis of artificial endonucleases. Methods as below:

[0070] (1) Add chelating agent

[0071] To PNA-lys-NH 2 Add Tris-HCl buffer solution with pH=5-8 to dissolve it, then add EDTA anhydride, stir and react at 30°C for 0.5-1h to obtain PNA-lys-NH-EDTA.

[0072] (2) Add cerium ion

[0073] Add Ce ions (Ce(NO 3 ) 4 Form), to obtain PNA-Ce complexes, that is, artificial endonucleases.

Embodiment 2

[0075] positioning cut

[0076] In this example, the ability of the 10-mers PNA-cerium complex artificial nuclease prepared in Example 1 to cleave 26-mers Oligo DNA in a targeted manner was tested.

[0077] If there is a complementary sequence or a homologous sequence part between the PNA single strand and the DNA single strand, it can form a hybrid double strand under annealing conditions through molecular hybridization. The 10 bases in the 10mers PNA-lys-NH-EDTA structure are complementary to the 10 bases in the 26-mers ODN (sequence 5'-TTCACTGTACGGATCGATTACTTTAG-3'), so under appropriate conditions, the bases can be complementary Pairing principle, hybridization forms a double helix structure, such as figure 2 shown.

[0078] The 26-mer oligonucleotide substrate and the artificial endonuclease of Example 1 were reacted in Tris-HCl buffer solution with pH=6-8 at a temperature of 30°C-40°C for 24-48 hours. Then the reaction product was taken for electrophoresis.

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Abstract

The invention provides the artificial inner cut enzyme which can cut phosphodiester bond in nucleic acid selectivitily. The inner cut enzyme contains recognition and cutting details. The recognition details is single strand of peptide nucleic acid which constitutional unit is 6-200 qae glycine. The cutting details is chelating agent of rare earth metal ion which is chelated with metal ion at the ammonia or carbon terminal of single strand of peptide nucleic acid and which has joint between the chelating agent and single strand. the artificial inner cut enzyme can be used implemental enzyme widely in molecular biology, genetic engineering and medical treatment area.

Description

technical field [0001] The invention relates to the fields of molecular biology and enzymology, and more specifically relates to an artificial endonuclease which selectively cuts phosphodiester bonds in nucleic acid containing a peptide nucleic acid recognition element and its preparation method and application. Background technique [0002] As we all know, nucleic acid is the carrier of genetic information and the material basis of gene expression. It plays a very important role in normal life activities such as the growth, development, and reproduction of organisms. On the other hand, nucleic acid is also closely related to abnormal conditions of life, such as the occurrence of tumors, radiation damage, and genetic diseases. Therefore, the research on nucleic acid has become an important topic in the research of modern molecular biology, medicine and chemical biology. Selectively cutting phosphodiester bonds in nucleic acids is one of the core technologies of genetic engi...

Claims

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Application Information

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IPC IPC(8): C07H21/00C12N9/22C12P19/34C12Q1/68
Inventor 沈鹤柏杨永桃
Owner SHANGHAI NORMAL UNIVERSITY
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