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Hairpin structure containing CpG site and monomolecular mechanical method for measuring influence of CpG adjacent sequences on protein dissociation time constant

A hairpin structure and site technology, applied in biochemical equipment and methods, measuring devices, analysis materials, etc., can solve problems such as unclearness and affecting the affinity of protein substrates, and achieve fast collection and improved accuracy Effect

Active Publication Date: 2020-06-12
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, how the sequence adjacent to the CpG site affects the substrate affinity of the protein is still unclear

Method used

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  • Hairpin structure containing CpG site and monomolecular mechanical method for measuring influence of CpG adjacent sequences on protein dissociation time constant
  • Hairpin structure containing CpG site and monomolecular mechanical method for measuring influence of CpG adjacent sequences on protein dissociation time constant
  • Hairpin structure containing CpG site and monomolecular mechanical method for measuring influence of CpG adjacent sequences on protein dissociation time constant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1: test group comprises the construction of the hairpin structure of CpG site

[0093] as per figure 1 The procedure shown produces hairpin structures.

[0094] 1 Preparation of biotin handle and digoxin handle

[0095] 1.1 Polymerase chain reaction of biotin modification / digoxigenin modification

[0096] Perform biotin-modified / digoxigenin-modified PCR on the pbluescrIISK+ plasmid by adding:

[0097] 1 μl of biotin / digoxigenin handle upstream primer at a concentration of 10 μM;

[0098] 1 μl of primers downstream of the biotin / digoxigenin handle with a concentration of 10 μM (both primers were purchased from Jinweizhi Company);

[0099] 1 μl of dATP with a concentration of 10 mM (Cat. No.: 4026Q, Baoriji Biotechnology Co., Ltd.);

[0100] 1 μl of dGTP with a concentration of 10 mM (Cat. No.: 4027Q, Baoriji Biotechnology Co., Ltd.);

[0101] 1 μl of dCTP with a concentration of 10mM (Cat. No.: 4028Q, Baoriyi Biotechnology Co., Ltd.);

[0102] 0.9 μl of...

Embodiment 2

[0181] Embodiment 2: Control group comprises the construction of the hairpin structure of CpG site

[0182] Compared with the construction of the hairpin structure of the CpG site of the test group, the control group needs to additionally synthesize the third nucleic acid fragment of the control group and the fourth nucleic acid fragment of the control group,

[0183] The annealing reaction system of the third nucleic acid fragment of the control group is:

[0184] 8 μl of 50 μM nucleic acid chain whose sequence is shown in SEQ ID NO.18;

[0185] 8 μl of 50 μM nucleic acid chain whose sequence is shown in SEQ ID NO.19;

[0186] 4 μl 5X DNA Annealing Buffer.

[0187] The annealing reaction system of the fourth nucleic acid fragment of the control group is:

[0188] 8 μl of 50 μM nucleic acid chain whose sequence is shown in SEQ ID NO.20;

[0189] 8 μl of 50 μM nucleic acid chain whose sequence is shown in SEQ ID NO.21;

[0190] 4 μl 5X DNA Annealing Buffer.

[0191] The t...

Embodiment 3

[0205] Example 3: Using single-molecule magnetic tweezers to measure the length change of the hairpin structure under the action of external force

[0206] Making the reaction pool: The reaction pool consists of a slide without holes and a cover glass with holes.

[0207] 1) Punch holes on the cover glass: take a 60mm×24mm glass slide, and use a TC-169 multi-function grinding and engraving machine to punch two holes as the water inlet and water outlet.

[0208] 2) Cleaning of glass slides: Place the coverslip and slide glass vertically in the antibody incubation box, add ultrapure water containing detergent, sonicate in water bath for 30 minutes, rinse with pure water 3 times; add isopropanol, water bath Ultrasonic for 30 minutes, rinse 3 times with pure water; add isopropanol, ultrasonically in water bath for 30 minutes, rinse 3 times with pure water; add pure water, ultrasonically in water bath for 30 minutes, rinse 3 times with pure water; finally add 75% ethanol to soak B...

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Abstract

The invention relates to a monomolecular mechanical method for measuring the influence of CpG adjacent sequences on a protein dissociation time constant. Based on a single-molecule mechanical method,dissociation events of the protein from multiple CpG sites on a same DNA molecule are detected in a high-throughput manner, and the influence of different adjacent sequences on the interaction betweenthe protein and the CpG sites is compared and analyzed. According to the invention, a hairpin structure containing the CpG sites is constructed; a stem part of the structure contains a plurality of CpG sites which are distributed at equal intervals; the CpG adjacent sequences, such as CG, GC, AT and TA, can be set as required, the corresponding CpG sites of the control DNA hairpin structure onlycontain one adjacent sequence, the dissociation time of the protein at each CpG site is compared and analyzed, and how the CpG adjacent sequences influence the protein dissociation time constant can be accurately measured. The method can be used for repeatedly detecting time and probability of the interaction between the protein and different CpG sites in real time in a high-throughput manner to obtain an accurate dissociation time constant, can be widely used for precisely analyzing the interaction between protein and nucleic acid, and is beneficial to life science and medicine research and development.

Description

technical field [0001] The invention belongs to the field of biophysics, and in particular relates to a hairpin structure including a CpG site and a single-molecule mechanics method for measuring the influence of CpG adjacent sequences on the time constant of protein dissociation. Background technique [0002] There are many proteins that bind to CpG sites, for example, TET1 (Ten-eleven translocationmethylcytosine dioxygenase 1, TET1) protein, also known as CXXC6, is one of the zinc finger structural proteins. Human TET1 protein is mainly composed of six structural domains, among which the CXXC domain is a zinc finger domain, which can bind to the CpG site with the participation of the cofactor zinc ion. TET1 can directly bind to the promoter region of the gene, catalyze the conversion of 5-methyl-cytosine into 5-hydroxymethyl-cytosine, recruit histone methyltransferase to bind to the promoter region, and promote gene transcription. However, how the sequence adjacent to the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113G01N33/53
CPCC12N15/113G01N33/5308C12N2310/531
Inventor 于仲波王泽瑜郑薇梁琳李旭
Owner NANKAI UNIV
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