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Eukaryote-derived Argonaute protein and application thereof

A eukaryotic and protein technology, applied in the fields of application, biochemical equipment and methods, plant genetic improvement, etc.

Active Publication Date: 2022-06-10
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] For a long time, people have been widely concerned about the important role of eAgo in the RNAi pathway, and eAgo with high RNase activity has been found in higher animals, plants and yeast cells, but The interaction between eukaryotic Argonaute and DNA has not been explored in detail. Although some studies have found that eukaryotic Argonaute protein can cleave RNA with single-stranded DNA as a guide, it has not found its cleaving activity on DNA.
At the same time, with regard to the application of Argonaute proteins in gene editing, since eukaryotic Argonaute proteins from higher animals and plants may participate in the RNAi pathway in recipient cells, effective gene editing cannot be achieved, so there is currently no use of eukaryotic Argonaute proteins for gene editing. edit

Method used

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  • Eukaryote-derived Argonaute protein and application thereof
  • Eukaryote-derived Argonaute protein and application thereof
  • Eukaryote-derived Argonaute protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Embodiment 1 TteAgo expression and purification

[0096] Transform the pET28a-CL7-TteAgo plasmid into Escherichia coli BL21(DE3), inoculate a single colony into LB liquid medium containing 50 μg / mL kanamycin, and culture it on a shaking table at 37°C and 220 rpm. When the OD600 When reaching 0.8, move to 18 ° C shaker, IPTG induction overnight. Collect the bacteria by centrifugation at 6000rpm for 10min, wash the bacteria with Buffer A (20mM Tris-HCl pH 7.4, 500mM NaCl, 10mM imidazole), resuspend the bacteria in Buffer A, add PMSF at a final concentration of 1mM, and crush under high pressure. Centrifuge at 18000rpm for 30min, and collect the supernatant. After the supernatant was filtered, Ni-NTA purification was performed. Wherein, the amino acid sequence of the CL7-TteAgo fusion protein is shown in SEQ ID NO.2, and the polynucleotide sequence of the CL7-TteAgo fusion protein is shown in SEQ ID NO.4.

[0097] Wash 10 column volumes with Buffer A containing 10 mM im...

Embodiment 2

[0099] The cleavage activity of embodiment 2TteAgo

[0100]In order to assess which combinations of RNA / DNA guides and RNA / DNA targets TteAgo was able to cleave, activity assays were performed on all possible combinations in this example.

[0101] Lysis assays were all performed at 37°C in a 5:2:1 (TteAgo:guide:target) molar ratio. Put 1uM TteAgo and 400nM guide in the solution containing 10mM HEPES-NaOH (pH 7.5), 100mM NaCl, 5mM MnCl 2 and 5% glycerol in reaction buffer and incubated at 37°C for 10 min for guide loading. Nucleic acid targets were added to a final concentration of 200 nM. After 1 h reaction at 37°C, the reaction was stopped by mixing the samples with 2x RNA loading dye (95% formamide, 18 mM EDTA, 0.025% SDS and 0.025% bromophenol blue) and heating at 95°C for 5 min. Lysates were resolved by 20% denaturing TBE-PAGE, stained with SYBR Gold (Invitrogen), and Gel Doc TM XR+ (Bio-Rad) visualization.

[0102] Figure 4 Figures A and B in the figure are schema...

Embodiment 3

[0104] Embodiment 3 The influence of guide molecule length on cutting effect

[0105] Referring to the experimental method in Example 2, RNA guides or DNA guides of different lengths were combined with TteAgo to verify its activity of cutting target RNA or target DNA.

[0106] Test results such as Figure 5 as shown, Figure 5 Figure A in the middle shows the guide-guided cleavage of target RNA by TteAgo within 30 minutes under the condition of 12-30nt 5’ phosphorylated RNA guide; Figure 5 Figure B in the middle shows the guide-guided cleavage of target RNA by TteAgo within 30 minutes under the condition of a 12-30nt 5’ phosphorylated DNA guide; Figure 5 Figure C in the middle shows that under the condition of 12-30nt long 5' phosphorylated RNA guide, TteAgo showed guide-guided target ssDNA cleavage within 60 minutes. from Figure 5 It can be seen that the RNA guide length ranges from 12-25nt and the DNA guide length ranges from 12-30nt, and the target RNA can be effecti...

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Abstract

The invention discloses an Argonaute protein derived from eukaryotes and application of the Argonaute protein. The amino acid sequence of the Argonaute protein is as shown in SEQ ID NO.1, or the amino acid sequence of the Argonaute protein has at least 50% of sequence consistency with the sequence as shown in SEQ ID NO.1; according to the invention, the specific cleavage activity of the eukaryotic Argonaute protein on DNA is proved for the first time, and experimental proof is provided for research on interaction between the eukaryotic Argonaute and DNA; meanwhile, the polypeptide, the nucleic acid, the expression vector, the composition, the kit and the method used in the invention can perform site-specific operation on intracellular and extracellular genetic materials, can be effectively applied to many fields of biotechnology, and provide a new tool for gene editing, modification and molecular detection based on the Argonaute polypeptide derived from eukaryotes.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to an Argonaute protein derived from eukaryotic organisms and an application thereof. Background technique [0002] Eukaryotic Argonaute (referred to as eAgo) plays a key role in the RNA interference (RNA interference, RNAi) pathway of eukaryotes, and is the main functional core of the RNA-induced silencing complex (RISC). Complementary RNA target-specific recognition guides small single-stranded RNA molecules to bind, either directly cleave the target through the intrinsic nuclease activity of some eAgos enzymes, or recruit other silencing proteins to act on the target, resulting in transcriptional repression. Thus, eAgos can regulate gene expression at the transcriptional level, protect hosts from RNA viruses, and maintain genome integrity by reducing transposon mobility. Recent studies have also shown that some AGO proteins can regulate gene expression thro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/55C12N15/113
CPCC12N9/22C12N15/113C12N2310/20
Inventor 马立新何如怡孙宝彤王飞王亚平李忠臣颜光波
Owner HUBEI UNIV
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