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Method for controlling intracellular mRNA positioning and translation process on basis of controllable phase separation liquid drops

A technology of translation process and phase separation, applied in chemical instruments and methods, DNA/RNA fragments, polypeptides containing positioning/targeting motifs, etc., can solve the problem of difficult manipulation of mRNA positioning, and achieve rapid and regional regulation of cells The effect of the function

Inactive Publication Date: 2020-07-31
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In response to the above problems, the present invention provides an important tool for controlling the positioning and translation process of mRNA in cells, so as to overcome the problem that it is difficult to manipulate mRNA positioning in cells now

Method used

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  • Method for controlling intracellular mRNA positioning and translation process on basis of controllable phase separation liquid drops
  • Method for controlling intracellular mRNA positioning and translation process on basis of controllable phase separation liquid drops
  • Method for controlling intracellular mRNA positioning and translation process on basis of controllable phase separation liquid drops

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Controlling the translation process of mRNA by using a control method based on controllable phase-separation liquid droplets to control the localization and translation process of mRNA in cells. Take the messenger RNA of CFP (cyan fluorescent protein) with FLAG tag as an example:

[0033] (1) Amplify the corresponding DNA fragment from the template plasmid containing the desired sequence by PCR. Add restriction enzyme sites to the amplified primers. Purified DNA fragments corresponding to the following proteins were recovered by DNA agarose gel electrophoresis and gel purification kits: FUSN, mCherry, PixE, PixD, FLAG, CFP, PP7, 4EBP1, GFP, tdPCP, etc. Among them, FUSN represents the N-terminal region of the FUS protein, which is a natural unstructured region IDR with phase separation ability, its amino acid sequence is shown in SEQ ID No: 5 in the sequence listing, and its corresponding DNA sequence is shown in SEQ ID No: 6 Shown; mCherry, CFP, GFP are red...

Embodiment 2

[0042] Example 2: A method for controlling intracellular mRNA localization and translation process based on controllable phase-separation droplets using fluorescence microscopy imaging method to visualize the translation process of mRNA. Take the recruitment of messenger RNA with CFP into droplets and light release as an example:

[0043] (1) Amplify the corresponding DNA fragment from the plasmid containing the template sequence by PCR. Add restriction enzyme sites to the amplified primers. Purified DNA fragments corresponding to the following proteins, FUSN, mCherry, PixE, PixD, CFP, PP7, GFP, tdPCP, MS2, tdMCP, etc., were recovered by DNA agarose gel electrophoresis and gel purification kit, wherein the DNA sequence of MS2 is as follows: SEQ ID No: 10; the amino acid sequence of tdMCP is shown in SEQ ID No: 11, and the corresponding DNA sequence is shown in SEQ ID No: 12.

[0044] The PCR amplification system used is as follows:

[0045]

[0046] (2) Obtain expression...

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Abstract

The invention discloses a method for controlling intracellular mRNA positioning and translation process on basis of controllable phase separation liquid drops. Protein liquid drops are constructed incells with a protein phase separation technology, an RNA aptamer is used for connecting mRNA, any specific mRNA can be called to the protein liquid drops, aggregation and disaggregation of the liquiddrops are controlled through an optogenetic technology, and calling and release of mRNA are realized. When the mRNA is wrapped in the liquid drops, translation of the mRNA is inhibited, the liquid drops are disaggregated in a specific illumination area, mRNA is released, the translation process is completed, so that accurate time and space control in the mRNA translation process in live cells is realized. The technology will be widely applied in the aspect of study of the influence of gene expression space regulation on cell migration and diseases in tissue development.

Description

technical field [0001] The invention relates to a precise time and space control method for the mRNA translation process in living cells, belonging to the fields of molecular biology and cell biology. Background technique [0002] The heterogeneous distribution of intracellular mRNA can ensure that intracellular life activities are carried out in different subcellular regions. Controlling intracellular mRNA localization and translation processes is a mechanism widely adopted by cells to control gene expression in time and space. This mechanism is especially pronounced in highly polarized asymmetric cells. Controlling the asymmetric localization of intracellular mRNA can make the translation product of mRNA—the protein—distribute in special subcellular compartments to exert local functions. The non-polar distribution of intracellular mRNA plays an important role in cell fate determination, cell migration, embryonic development and other processes. For example, in budding y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/62
CPCC07K14/47C07K2319/01C12N15/85
Inventor 孙育杰张宏晨邵世鹏
Owner PEKING UNIV