A prokaryotic-derived mbp_argonaute protein and its application

A protein and complex technology, applied in the field of molecular biology, can solve the problems of high price of chemically synthesized dsRNA, time-consuming preparation, non-specific gene suppression, no targeted cleavage, etc., and achieve strong cleavage activity, convenient design, and cost saving Effect

Active Publication Date: 2022-07-26
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no pAgos protein in the prior art that can effectively target and cut various types of RNA under normal temperature conditions and can be applied to RNA editing in animal and plant cells
The general RNAi technology requires the use of double-stranded RNA (dsRNA). The price of chemically synthesized dsRNA is high and the customization period is long. The price of in vitro transcription of dsRNA is relatively low, but the operation is difficult and time-consuming. Time-consuming preparation, non-specific gene suppression, etc.
As for CRISPR-based technology, it also needs to use long RNA guides, which have the same problems as RNAi technology. In addition, CRISPR-related proteins (such as Cas13a) rely on special motifs near the target site to recognize and bind to the target, which limits the potential The scope of editing, and CRISPR-associated proteins also have extremely strong non-specific "incidental cleavage" activity, which is worrying about its possible off-target response

Method used

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  • A prokaryotic-derived mbp_argonaute protein and its application
  • A prokaryotic-derived mbp_argonaute protein and its application
  • A prokaryotic-derived mbp_argonaute protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 MbpAgo expression and purification

[0053] The nucleotide sequence shown in SEQ ID NO: 2 was amplified from the cryogenic prokaryote Mucilaginibacter paludis, and was connected to pET28a by conventional methods to obtain the pET28a-MbpAgo plasmid, which was then transformed into E. coli Rosetta (DE3), and a single colony was obtained. It was inoculated into LB liquid medium containing 50 mg / mL kanamycin, and cultured in a shaker flask at 37 °C and 220 rpm on a shaker. 600 When it reached 0.8, it was moved to a shaker at 18°C ​​and induced with IPTG overnight. The cells were collected by centrifugation at 6000 rpm for 10 min, washed with Buffer A (20 mM Tris-HCl pH 7.4, 500 mM NaCl, 10 mM imidazole), and then resuspended in Buffer A, added with a final concentration of 1 mM PMSF, and disrupted by high pressure. Centrifuge at 18,000 rpm for 30 min, and collect the supernatant. After supernatant filtration, Ni-NTA purification was performed.

[0054] 20mM imi...

Embodiment 2

[0056] Example 2 MbpAgo cleavage activity assay

[0057] To assess which combinations of guide RNA / DNA and target RNA / DNA MbpAgo is able to cleave, activity assays were performed for all possible combinations. The sequence diagrams of target DNA, target RNA, guide ssDNA and guide ssRNA are as follows Figure 4 shown, where arrows indicate predicted cleavage sites

[0058] Cleavage experiments were all performed at 37°C in a 4:2:1 (MbpAgo:guide:target) molar ratio. Place 800 nM MbpAgo with 400 nM guide in a solution containing 10 mM HEPES-NaOH, pH 7.5, 100 mM NaCl, 5 mM MnCl 2 Mix with 5% glycerol in reaction buffer and incubate at 37°C for 10 min for guide loading. Nucleic acid targets were added to 200 nM. After 1 h of reaction at 37°C, the reaction was terminated by mixing the samples with 2x RNA loading dye (95% formamide, 18 mM EDTA and 0.025% SDS and 0.025% bromophenol blue) and heating at 95°C for 5 minutes. Lysates were resolved by 20% denaturing PAGE, stained with...

Embodiment 3

[0060] Example 3 Effect of gDNA length on target RNA cleavage activity

[0061] DNAs with a length of 8-40 nt were selected as guide DNAs, which were incubated with MbpAgo to form pAgo complexes, and the activities of different length guide DNAs on MbpAgo recognition and cleavage of target RNA were determined. The results are as Figure 7 shown.

[0062] The results show that the length of the guide DNA has a certain influence on the activity of MbpAgo to recognize and cut the target RNA. When the length of the guide DNA is in the range of 8-40nt, preferably 10-30nt, the target RNA can be effectively cleaved.

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Abstract

The present invention discloses a prokaryotic-derived Mbp_Argonaute protein and an application thereof, wherein the amino acid sequence of the Mbp_Argonaute protein is such as SEQ ID NO: 1 or a sequence with at least 50% or at least 80% homology with SEQ ID NO: 1 shown. The invention synthesizes an Argonaute protein gene derived from the cold-resistant prokaryote Mucilaginibacter paludis, and the protein is named as MbpAgo. It is found that the MbpAgo has binding activity to single-stranded guide DNA and is complementary to the target RNA paired with the single-stranded guide DNA. And / or the target DNA has nuclease activity, so the MbpAgo can be used to perform targeted RNA editing in vitro and in vivo, thereby performing specific site modification on genetic materials. The MbpAgo can not only modify the RNA of high-level structure, but also does not affect the endogenous RNAi pathway of animal and plant cells, providing a new powerful tool for RNA editing, and has strong cutting activity and good specificity.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a prokaryotic-derived Mbp_Argonaute protein and its application. Background technique [0002] At present, the eukaryotic Argonaute protein (referred to as eAgos) can catalyze the RNA-guided (gRNA)-guided RNA cleavage reaction at room temperature, and play a crucial role in the RNA interference (RNAi) pathway in vivo . Argonaute proteins derived from prokaryotes (pAgos for short) are more diverse in function and structure than eAgos, but their physiological functions have long been elusive. Early studies mainly focused on pAgos derived from high-temperature organisms, except that MpAgo derived from Marinitoga piezophila prefers to use 5'-terminal hydroxylated (5'OH) guide RNA (gRNA) to cleave target single-stranded DNA (ssDNA) and target In addition to RNA, the remaining pAgos derived from high temperature prefer to use 5'-terminal phosphorylated (5'P) gDNA to cleave ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/22C12N15/55C12N15/113
CPCC12N9/22C12N15/113C12N2310/20C12N2310/14Y02A50/30
Inventor 马立新李文强王飞何如怡刘洋
Owner HUBEI UNIV
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