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Protein proximity labeling method based on DNA nanotechnology and application thereof

A labeling method and nanotechnology technology, applied in the field of adjacent labeling, can solve the problems of affecting the function of the target protein, locating interactomics, affecting the accuracy and specificity of mass spectrometry identification, and complex operation, so as to improve the labeling efficiency and labeling efficiency. High and specific effect

Active Publication Date: 2022-06-07
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current proximity labeling technology needs to first fuse the enzyme of the proximity label with the target protein, and then transfect it into the cell for protein labeling. The operation is more complicated, expensive, and time-consuming, and the fusion may also affect the function of the target protein. , localization and even its interactomics
At the same time, during the process of expression and accumulation of engineered enzymes in cells, biotin in cell culture medium will be used to produce higher background labeling of adjacent proteins, which will affect the accuracy and specificity of mass spectrometry identification
However, there are no reports on the application of DNA nanotechnology to the proximity labeling of cell surface membrane proteins

Method used

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  • Protein proximity labeling method based on DNA nanotechnology and application thereof
  • Protein proximity labeling method based on DNA nanotechnology and application thereof
  • Protein proximity labeling method based on DNA nanotechnology and application thereof

Examples

Experimental program
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Embodiment 1

[0033] Embodiment 1 A protein proximity labeling method based on DNA nanotechnology, comprising the following steps:

[0034] (1) Synthesis of DNA nanostructures: The designed DNA sequences S1 (SEQ ID NO:1), S3 (SEQ ID NO:3) and S4 (SEQ ID NO:4) were mixed in TE buffer (10mmol / LTris- HCl; 1 mmol / L EDTA; pH 7.8-8.2), the DNA sequences S2 (SEQ ID NO: 4) and S5 (SEQ ID NO: 5) were mixed in equal proportions in TE buffer (10 mmol / LTris-HCl; 1 mmol at the same time) / L EDTA; pH 7.8-8.2), then put the two sequences into the PCR synthesizer respectively, annealed at 95°C for 10 minutes, then rapidly cooled from 95°C to 4°C, and finally kept at 4°C, and finally the single-stranded S1, S3 and S4 synthesize DNA nanostructure A, and single-stranded S2 and S5 synthesize DNA nanostructure B. Details of DNA sequences S1-S5 are shown in Table 1:

[0035]Table 1 DNA sequences

[0036]

[0037] During the synthesis of DNA nanostructures, agarose gel electrophoresis was used to verify whe...

Embodiment 2

[0044] Example 2 A protein proximity labeling method based on DNA nanotechnology and its immunoblotting and mass spectrometry identification

[0045] The synthetic steps of step (1)-(4) are identical with embodiment 1;

[0046] (5) Lyse cells to extract membrane proteins: use Mem-PER TM The Plus Membrane Protein Extraction Kit (purchased from ThermoFisher Scientific, Cat. No. 89842) was used to extract membrane proteins. Specifically, 1 mL of permeabilization buffer was added, and the cells were lysed at 4°C for 10 minutes. After centrifugation to remove the supernatant, 500 uL was added to the pellet for solubilization. buffer, incubated at 4°C for 30 minutes, and the supernatant obtained after centrifugation was the cell membrane protein.

[0047] (6) Enrichment of target protein: The target protein labeled with biotin was enriched with streptavidin-coupled magnetic beads (purchased from BeaverBio, Item No. 22307).

[0048] (7) Western blotting: add 20uL of 1× protein loa...

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Abstract

The invention belongs to the technical field of proximity labeling, and particularly relates to a protein proximity labeling method based on a DNA nanotechnology and application of the protein proximity labeling method. The DNA nanotechnology is utilized to target the enzyme to the immune synaptic part, compared with the current proximity labeling reaction, the method is simple and convenient to operate, economical, efficient and short in consumed time, the biotinylation proximity labeling can be completed only in 10 minutes, the enzyme of the proximity labeling does not need to be fused with the target protein in advance, transfection is not needed, and the cost is low. The method can be used for researching weak or instantaneous interaction between the proteins, can also be used for mass spectrum identification and proteomics analysis and mining of new protein molecules related to immune synapse, and provides a new thought for research and development of drugs. Meanwhile, by applying the DNA nanotechnology, signals can be effectively amplified, and the marking efficiency is greatly improved. Moreover, the specificity of the marker is good, and almost no non-specific background signal exists.

Description

technical field [0001] The invention belongs to the technical field of proximity labeling, in particular to a protein proximity labeling method based on DNA nanotechnology and its application. Background technique [0002] The strategy of proximity labeling technology is usually to fuse and express the adjacently labeled enzyme with the target protein, and then target the enzyme to a specific part of the cell by transfection of fusion constructs or alternative induction methods (such as virus infection, etc.), and then add biotin, etc. Small molecule substrate, the adjacent labeled enzyme can catalyze the covalent binding of the protein adjacent to the target protein in space with small molecules such as biotin, so that the target protein and the adjacent protein are biotinylated, and then coupled with streptavidin. The biotin-labeled proteins were enriched by coupled magnetic beads, and the proteins were identified and bioinformatically analyzed by mass spectrometry. At pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/16C12N5/00G01N33/533G01N33/68C12Q1/6804
CPCC12N9/22C12N5/0006G01N33/533G01N33/6848C12Q1/6804C12Q2563/131C12Q2563/155Y02A50/30
Inventor 张元庆郑紫蔚陈亮
Owner SUN YAT SEN UNIV
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