VbAgo RNA Editing via ssDNA Guide Specificity
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Solution Overview
Problem
Current RNA editing technologies face limitations, including pAgos with low RNA target cleavage activity under mesophilic conditions, DNA cleavage activity causing DNA damage, costly and time-consuming dsRNA synthesis, long-lasting gene interference effects, and non-specific CRISPR-associated protein collateral cleavage.
Innovation Solution
A mesophilic prokaryotic Argonaute protein, VbAgo, with specific RNA target cleavage activity, derived from Verrucomicrobia bacterium, which binds to single-stranded guide DNA and exhibits nuclease activity only on complementary RNA targets, allowing for efficient and specific RNA editing in vivo and in vitro.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If pAgos from thermophilic organisms are used for RNA target cleavage, then cleavage activity can be achieved, but the activity level is low under mesophilic conditions which limits application
Solution Approach 1:
The patent changes the source organism parameter from thermophilic to mesophilic prokaryotes, thereby adapting the pAgo enzyme's optimal activity temperature to mesophilic conditions (37°C) while maintaining high RNA target cleavage activity. This parameter change resolves the contradiction by enabling reliable cleavage activity under physiologically relevant temperatures.
2Ease of operation
If eAgos are used for RNA cleavage, then RNA editing can be performed, but they may interfere with the RNAi function of the cell itself
Solution Approach 1:
The patent uses prokaryotic Argonaute proteins as a functional copy of eukaryotic Argonaute proteins, but from a different evolutionary lineage that lacks RNAi pathways. This copying approach allows RNA editing functionality to be introduced without the harmful side effect of interfering with eukaryotic RNAi mechanisms, as prokaryotes simply do not possess these pathways.
3Adaptability or versatility
If CRISPR-Cas nucleases are used for RNA targeting, then RNA can be targeted, but costly and time-consuming gRNA preparation is required
Solution Approach 1:
The patent employs short ssDNA guides instead of traditional CRISPR gRNAs, which can be synthesized more quickly and cheaply. The simplified guide molecule design reduces preparation complexity and cost while maintaining effective RNA targeting capability through the pAgo enzyme system.
4Ease of operation
If pAgos with DNA cleavage activity are used, then RNA editing can be performed, but DNA damage may occur
Solution Approach 1:
The patent extracts and isolates the RNA-specific cleavage function from pAgo enzymes that possess both DNA and RNA cleavage activities. By selecting and characterizing pAgos that specifically target RNA or have predominant RNA cleavage activity, the harmful DNA cleavage function is effectively removed or minimized, enabling safe RNA editing without genotoxicity concerns.
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
VbAgo provides a novel, cost-effective, and highly specific tool for RNA editing, avoiding DNA cleavage and off-target effects, with improved cleavage efficiency and reduced synthesis costs, suitable for animal and plant cells.
Implementation Method 1
binds to single-stranded guide DNA and exhibits nuclease activity only on complementary RNA targets
Implementation Method 2
exhibits nuclease activity only on complementary RNA targets
Data Source
AI summary
An application of a prokaryotic Argonaute protein with only a cleavage activity of a target ribonucleic acid (RNA) in RNA editing is provided. The Argonaute protein is derived from a mesophilic prokaryotes Verrucomimicrobia bacterium, and its amino acid sequence is shown in SEQ ID NO: 1 or a protein with high similarity to SEQ ID NO: 1 and the same function. This protein has binding activity to a single-stranded guide DNA and nuclease activity only to the target RNA complementarily paired with the single-stranded guide DNA. Therefore, the protein can be utilized for in vitro and in vivo targeted RNA editing, providing a new powerful tool for RNA editing.