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Prokaryote-derived Mbp_Argonaute protein and application thereof

A prokaryotic and protein technology, applied in the field of molecular biology, can solve the problems of time-consuming, no targeted cleavage, and high price of chemically synthesized dsRNA

Active Publication Date: 2021-09-10
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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  • Prokaryote-derived Mbp_Argonaute protein and application thereof
  • Prokaryote-derived Mbp_Argonaute protein and application thereof
  • Prokaryote-derived Mbp_Argonaute protein and application thereof

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 cold-resistant prokaryote Mucilaginibacter paludis, and connected to pET28a by conventional methods to obtain the pET28a-MbpAgo plasmid, and then transformed into Escherichia coli Rosetta (DE3), single colony Inoculate into LB liquid medium containing 50 mg / mL kanamycin, shake the flask on a shaker at 37°C and 220 rpm, when the OD of the bacteria 600 When it reaches 0.8, move to a shaker at 18°C ​​and induce IPTG 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.

[0054] Wash 10 column volumes each with 2...

Embodiment 2

[0056] Example 2 Determination of MbpAgo cleavage activity

[0057] To assess which combinations of guide RNA / DNA and target RNA / DNA were able to be cleaved by MbpAgo, activity assays were performed for all possible combinations. The sequence diagram of target DNA, target RNA, guide ssDNA and guide ssRNA is as follows Figure 4 , where the arrow indicates the predicted cleavage site

[0058] The cleavage assays were all performed at 37°C with a 4:2:1 (MbpAgo:guide:target) molar ratio. Put 800nM MbpAgo with 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 20OnM. 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 and 0.025% SDS and 0.025% bromophenol blue) and heating at 95°C for 5 min. The lysate was resolved by 20% denaturing PAGE, stained w...

Embodiment 3

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

[0061] DNAs with a length of 8-40nt were selected as guide DNAs, incubated with MbpAgo to form pAgo complexes, and the activity of guide DNAs with different lengths to recognize and cut target RNA by MbpAgo was measured. Measurement results such 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, wherein when the length of the guide DNA is in the range of 8-40nt, preferably 10-30nt, the target RNA can be effectively cut.

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Abstract

The invention discloses a prokaryote-derived Mbp_Argonaute protein and application thereof. The Mbp_Argonaute protein has an amino acid sequence shown in SEQ ID NO: 1 or as shown in a sequence which has at least 50% or at least 80% of homology with SEQ ID NO: 1. According to the invention, an Argonaute protein gene derived from a cold-resistant prokaryotic organism Mucilaginibacter paaluis is synthesized, the protein is named as MbpAgo, and researches find that the MbpAgo has binding activity to single-stranded guide DNA and has nuclease activity to target RNA and / or target DNA complementarily paired with the single-stranded guide DNA, therefore, the MbpAgo can be used for targeted RNA editing in vivo and in vitro, and then specific site modification of genetic material. The MbpAgo not only can modify RNA with a high-grade structure, but also does not influence an endogenous RNAi pathway of animal and plant cells, provides a brand new powerful tool for RNA editing, and is high in cutting activity and good in specificity.

Description

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

Claims

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Application Information

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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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