MbpAgo Prokaryotic Argonaute for Room-Temperature RNA Cleavage
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Solution Overview
Problem
Current RNA editing technologies, particularly those involving prokaryotic Argonaute proteins, face limitations in effectively cleaving various types of RNAs at room temperature and applying to plant and animal cells, with existing methods being costly, time-consuming, and non-specific, and lacking the ability to target highly-structured RNAs without interfering with endogenous RNAi pathways.
Innovation Solution
The development of MbpAgo, an Argonaute protein from the psychrotolerant prokaryote Mucilaginibacter paludis, which exhibits binding activity for single-stranded guide DNA and nuclease activity for target RNA/DNA, enabling site-specific modification of genetic material without affecting endogenous RNAi pathways in plant and animal cells, and allowing for the cleavage of highly-structured RNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If eAgos are used to cleave target RNA, then cleavage activity is achieved, but endogenous RNAi pathways are interfered with
Solution Approach 1:
The patent divides the Argonaute protein source into distinct segments: eukaryotic Argonautes (eAgos) that interfere with RNAi pathways versus prokaryotic Argonautes (pAgos) that do not. By selecting pAgos from psychrotolerant prokaryotes, the invention segments the problem source to eliminate harmful interference while preserving beneficial RNA cleavage activity.
Solution Approach 2:
The patent introduces an intermediary solution by using prokaryotic Argonaute proteins as mediators between the guide DNA and target RNA. These pAgos serve as alternative enzymes that can perform RNA cleavage without activating or interfering with the eukaryotic RNAi pathway, thus mediating the desired effect while avoiding harmful side effects.
2Reliability
If thermophilic pAgos are used, then gDNA-guided cleavage activity is achieved, but activity at mesophilic temperatures is low
Solution Approach 1:
The patent applies parameter changes by selecting pAgos from psychrotolerant (cold-adapted) prokaryotes instead of thermophilic ones. This changes the optimal temperature parameter of the enzyme, shifting its activity peak from high temperatures to room temperature and mesophilic ranges, thereby resolving the temperature mismatch problem.
3Adaptability or versatility
If CRISPR/Cas nucleases are used, then programmable RNA cleavage is achieved, but long gRNA transcription and purification is time-consuming
Solution Approach 1:
The patent extracts the essential function of CRISPR/Cas systems (programmable RNA cleavage) while eliminating the cumbersome components. By using pAgos that directly bind short gDNA guides without requiring long gRNA transcripts or complex purification steps, the invention extracts the core functionality and removes the time-consuming elements.
Solution Approach 2:
The patent employs short, synthetic gDNA guides that can be directly used without extensive purification. These short nucleic acid guides are simple, inexpensive to synthesize, and do not require the complex transcription and purification protocols needed for long gRNA molecules in CRISPR/Cas systems.
4Temperature
If NgAgo is used, then room temperature RNA cleavage is achieved, but cleavage site is uncertain and highly-structured RNA cannot be cleaved
Solution Approach 1:
The patent enhances universality by discovering pAgos from psychrotolerant prokaryotes that combine multiple functions: they work at room temperature like NgAgo, but additionally provide precise cleavage site determination and the ability to cleave highly-structured RNAs. This multi-functional pAgo system resolves the limitations of NgAgo.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
MbpAgo provides a powerful tool for RNA editing with high cleavage activity and specificity, reducing the need for expensive and time-consuming dsRNA synthesis, avoiding off-target effects, and enabling efficient RNA editing across different RNA types, including highly-structured RNAs, at room temperature.
Implementation Method 1
binding activity for single-stranded guide DNA
Implementation Method 2
nuclease activity for the target RNA and target DNA complementarily paired with the single-stranded guide DNA
Data Source
AI summary
Mbp_Argonaute proteins from prokaryotes and application thereof are provided. The Mbp_Argonaute protein consists of an amino acid sequence as shown in SEQ ID NO: 1 or a sequence with at least 50% or at least 80% of homology with the amino acid sequence as shown in SEQ ID NO: 1. An Argonaute protein gene derived from a cold-resistant prokaryote Mucilaginibacter paaluis is synthesized and named as MbpAgo, which 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, the MbpAgo can be used for the target RNA editing in vivo and in vitro to achieve site-specific modification of genetic material. The MbpAgo can modify highly-structured RNAs and not affect an endogenous RNAi pathway of animal and plant cells, provides a new and powerful tool for RNA editing with high cleavage activity and good specificity.