Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Prokaryote-derived argonaute protein and application thereof

A prokaryotic and protein technology, applied in the field of molecular biology, can solve problems such as short synthesis cycle, and achieve the effects of short synthesis cycle, cost saving and good specificity

Active Publication Date: 2021-03-23
HUBEI UNIV
View PDF3 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006]The significance of solving the above problems and defects is: it is generally believed that pAgos may not interfere with the RNAi pathway in animal and plant cells, and that DNA guides have a shorter and cheaper cycle than RNA synthesis, and pAgos Recognition and binding of target RNA without relying on specific motifs near the target site, which enables editing of arbitrary sites in RNA

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Prokaryote-derived argonaute protein and application thereof
  • Prokaryote-derived argonaute protein and application thereof
  • Prokaryote-derived argonaute protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1 KmAgo expression and purification

[0108] Transform the pET28a-KmAgo plasmid into Escherichia coli BL21(DE3), inoculate a single colony into LB liquid medium containing 50 mg / mL kanamycin, and culture it on a shaker at 37°C and 220 rpm. When the OD600 of the bacteria reaches At 0.8, move to a shaker at 18°C ​​overnight. Collect the cells by centrifugation at 6000 rpm for 10 min, wash with Buffer A (20 mM Tris–HCl pH 7.5, 500 mM NaCl, 10 mM imidazole), resuspend the cells in Buffer A, add PMSF at a final concentration of 1 mM, and press broken. Centrifuge at 18000 rpm for 30 min and collect the supernatant. After the supernatant was filtered, Ni-NTA purification was performed.

[0109] Wash 9 column volumes with 10 mM imidazole and 20 mM imidazole (add in 3 times), wash 3 column volumes with 50 mM, 80 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 1 M, and take samples Carry out SDS-PAGE detection. Collect the eluted fraction containing the high-purity targe...

Embodiment 2

[0111] Example 2 Testing the cleavage activity of KmAgo

[0112] To assess which combinations of RNA / DNA guides and RNA / DNA targets KmAgo was able to cleave, activity assays were performed for all possible combinations. Cleavage assays were all performed at 37 °C at a 4:2:1 (pAgo:guide:target) molar ratio. Place 800 nM KmAgo with 400 nM guide in a solution containing 10 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 5 mM 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 terminated by mixing the sample 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 with SYBR Gold (Invitrogen) and stained with Gel Doc TM XR+ (Bio-Rad) visualization.

[0113] The product band (34 nt) was not observed in DNA / RNA (guide / target) control assays ...

Embodiment 3

[0115] Example 3 KmAgo can be used at extremely low Mn 2+ cleavage of target RNA

[0116] Subsequent activity assays focused on finding the Mn for which KmAgo exhibits guide-directed target RNA cleavage within 15 min 2+ concentration range. mn 2+ The concentration range is set to 0-10 mM, and when the guide is 5’ phosphorylated DNA, the target RNA can be cut efficiently when the Mn2+ concentration is 0.01 mM ( Figure 7 ); when the guide is 5’ phosphorylated RNA, when the concentration of Mn2+ is greater than 0.5 mM, the target RNA can be effectively cut ( Figure 8 ).

[0117] Figure 7 DNA guides were incubated with KmAgo for 10 minutes at room temperature before adding the target. A 34 nt product band appeared between 0.05-10 mM, indicating an efficient reaction.

[0118] Figure 8 RNA guides were incubated with KmAgo for 10 min at room temperature prior to addition of target. A 34 nt product band appeared between 0.05-10 mM, indicating an efficient reaction.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the technical field of molecular biology, and discloses prokaryote-derived argonaute protein and application thereof. The prokaryote-derived argonaute protein is derived frommedium-temperature prokaryote kurthia masiliensis, and the prokaryote-derived argonaute protein is named as KmAgo. According to the protein and the application, polypeptide, nucleic acid, an expression vector, a composition, a kit and a method can perform site-specific modification on intracellular and extracellular genetic substances so that the protein can be effectively applied to many fields of biotechnology, such as nucleic acid detection, gene editing and gene modification, and a new tool is provided for gene editing, modification and molecular detection of the prokaryote-derived Argonaute polypeptide.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to an Argonaute protein derived from a prokaryote 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 key 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 RNA guides (gDNA) that utilize 5' terminal hydroxylation (5'OH) to cleave target single-stranded DNA (single-stranded DNA, ssDNA) and target Except for RNA, pAgos from other high-temperature sources prefer to us...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N9/22C12N15/55C12P19/34C12N15/63C12N15/867
CPCC12N9/22C12P19/34C12N15/63C12N15/86C12N2740/15043C12N2740/10043
Inventor 马立新王亚平刘洋姜小满
Owner HUBEI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com