Polypeptide probe for recognizing G-quadruplex and application of polypeptide probe in detection of G-quadruplex in cells

A quadruplex and probe technology, applied in the field of biological probes, can solve the problems of inability to detect G-quadruplexes, inability to detect G-quadruplexes in cells, etc., and achieve high affinity and selectivity and simple structure. , the effect of small interaction

Pending Publication Date: 2020-09-11
长治医学院
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to overcome the deficiencies in the prior art, one of the purposes of the present invention is to provide a polypeptide probe for identifying G-quadruplexes, which solves the problem that existing probes cannot be applied to the technology of detecting G-quadr

Method used

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  • Polypeptide probe for recognizing G-quadruplex and application of polypeptide probe in detection of G-quadruplex in cells
  • Polypeptide probe for recognizing G-quadruplex and application of polypeptide probe in detection of G-quadruplex in cells
  • Polypeptide probe for recognizing G-quadruplex and application of polypeptide probe in detection of G-quadruplex in cells

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Preparation of G4P.

[0043] Insert the G4P gene sequence between the Nde I and EcoR I restriction sites of the pet28b vector, and transform it into BL21-DE3 expression bacteria through plasmids. When the bacteria are cultured to OD 0.5, add 0.8mM final concentration of IPTG, and induce 4 Hours, then protein extraction, purification, protein extraction using Capturem TM His-Tagged Purification Miniprep Kit (TAKARA). The purified protein was stored in a solution: 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1 mM EDTA, 50% glycerol at -20°C.

Embodiment 2

[0045] Validation of the specificity of G4P recognition of G-quadruplexes.

[0046] In this example, the enzyme-linked immunosorbent assay was used to determine the binding ability of G4P to 21 G-quadruplexes of different sequences and several non-G-quadruplex control nucleic acids. The specific method is: heat 21 biotinylated oligonucleotides (see Table 1 for the sequence) in a buffer containing 10mM Tris-HCl (pH 7.4) and 150mM KCl to 95°C, then slowly anneal and cool to 20°C . The annealed oligonucleotides were immobilized on streptavidin-coated multiwell plates (Sigma-Aldrich), and then incubated with corresponding concentrations of G4P or RHAU23. Using anti-FLAG mouse monoclonal antibody (TransgenBiotech, China), HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Transgen Biotech, China) and TMBELISA substrate (Transgen Biotech, China), according to the manufacturer's manual to operate. Absorbance at 450 nm was measured on a Multi-PlateReader (Biotek, USA). Dis...

Embodiment 3

[0056] G4P recognizes G-quadruplexes in cells.

[0057] In order to identify G-quadruplexes in cells, the present invention expresses G4P in human A549 cells and uses it to carry out co-immunoprecipitation of G-quadruplexes. After the G4P-bound DNA library is constructed, next-generation sequencing is performed, and then the DNA sequence in the sequencing result is matched to the corresponding site in the genome.

[0058] The main steps are as follows:

[0059] Construction of G4P-ChIP plasmid:

[0060] The DNA encoding G4P was synthesized at Generay Biotechnology (Shanghai, China), and the DNA sequence was inserted between the NheI and EcoRI sites of the pIRES2-EGFP vector to obtain pG4P-IRES2-EGFP. The coding sequence of the nuclear localization signal peptide (NLS: PKKKRKV) of the SV40 major antigen was synthesized in Sangon (Shanghai, China), and inserted into the Nhe I site of pG4P-IRES2-EGFP to obtain the plasmid pNLS-G4P-IRES2-EGFP.

[0061] A DNA fragment expressing...

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Abstract

The invention discloses a polypeptide probe for recognizing G-quadruplex and application of the polypeptide probe in detection of the G-quadruplex in the cells. The polypeptide probe for recognizing the G-quadruplex contains 2-4 G-quadruplex combined structural domains and linking peptides for connecting the adjacent G-quadruplex combined structural domains, wherein each G-quadruplex combined structural domain contains one specific structural region, and an amino acid sequence of the specific structural region is PGHLKGREIGMWY (SEQ ID NO.1). The polypeptide probe provided by the invention is strong in specificity, is compatible with reducing environments in the cells and can be applied to the detection of the G-quadruplex in the cells.

Description

technical field [0001] The invention belongs to the technical field of biological probes, in particular to a polypeptide probe for identifying G-quadruplexes and its application in detecting G-quadruplexes in cells. Background technique [0002] G-quadruplexes are four-stranded higher order structures formed by guanine-rich nucleic acids. Potential G-quadruplex-forming sequences (PQS) are extremely abundant in the genomes of higher animal cells, and the detection and quantification of G-quadruplexes in the genome is very important for studying the biological functions of G-quadruplexes. Although G-quadruplexes are readily formed in single-stranded nucleic acids in vitro, the environment in cells is more complex. For example, guanine-rich sequences in chromosomes are restricted to long double-stranded DNA, and two complementary DNA strands pair up with each other. In addition, the DNA in the nucleus is bound and compressed by a large number of proteins. These conditions ar...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12Q1/6869
CPCC07K14/00C12Q1/6869C12Q2535/122C12Q2531/113C12Q2537/165C07K7/06C07K2319/80C07K2319/85C07K14/47C12Q2565/133C12Q1/68
Inventor 谭铮郑克威郑金平
Owner 长治医学院
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