Genetically Encodable Guide RNA for Endogenous ADAR2 Recruitment
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Solution Overview
Problem
Existing methods for introducing targeted point mutations into RNA using ADAR enzymes are inefficient as they require modified enzymes and guide RNAs with chemically altered nucleotides, necessitating in vitro production and transfection, which is not genetically encodable or cost-effective.
Innovation Solution
Designing genetically encodable guide RNAs with specific nucleotide segments that recruit endogenous ADAR enzymes to introduce targeted point mutations, allowing for efficient and reversible editing of RNA without altering the coding gene, using endogenous enzymes to introduce point mutations directly into RNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If modified ADAR enzymes with SNAP tag or In peptide are used to direct enzyme activity to new substrates, then targeted RNA editing can be achieved, but the device complexity increases and endogenous enzymes cannot be utilized
Solution Approach 1:
The invention extracts and utilizes the endogenous ADAR2 enzyme's natural deaminase domain, separating it from the need for complex modifications like SNAP tags or In peptides. The guide RNA alone directs the naturally occurring enzyme to target substrates, simplifying the overall system while maintaining editing precision.
Solution Approach 2:
The invention enables the cell's own endogenous ADAR2 enzymes to perform the editing function without requiring exogenous modified enzymes. The guide RNA recruits the cell's natural enzymes to carry out the targeted deamination, making the system self-sufficient and reducing complexity.
2Reliability
If guide RNA contains chemically altered nucleotides such as benzylguanine, then guide RNA can be activated and bind to enzyme, but the guide RNA cannot be genetically encodable and requires in vitro production
Solution Approach 1:
The invention changes the nucleotide composition parameters of the guide RNA to use only naturally occurring, genetically encodable nucleotides. This allows the guide RNA to be transcribed directly from DNA templates in living cells, eliminating the need for complex in vitro chemical synthesis while maintaining binding reliability through optimized sequence design.
3Manufacturing precision
If chemically altered nucleotides are used in guide RNA, then enzyme binding can be achieved, but transfection into cells is required which reduces efficiency
Solution Approach 1:
The system uses endogenous ADAR2 enzymes already present in the cell, eliminating the need for transfection of modified enzymes. The guide RNA, being genetically encodable, can also be delivered via simpler methods or expressed from integrated DNA, significantly improving delivery efficiency while maintaining editing precision.
4Productivity
If endogenous ADAR2 enzymes are used with genetically encodable guide RNAs, then the method becomes cost-effective and efficient, but the guide RNA structure must be precisely designed
Solution Approach 1:
The guide RNA is divided into functionally distinct segments: a deaminase recruitment element (DRE) that binds ADAR2, a spacer region for structural stability, and a target-complementary region for specific mRNA binding. This segmentation allows each element to be optimized independently, achieving high productivity while managing structural complexity through modular design.
Solution Approach 2:
The guide RNA structure incorporates universal elements that work across different target sequences. The DRE and spacer regions provide universal structural and functional properties, while only the target-complementary segment needs to be customized, reducing overall design complexity while maintaining high editing productivity for various targets.
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
This approach enables precise and efficient introduction of point mutations, reducing the need for exogenous proteins and chemical synthesis, enhancing therapeutic potential by using endogenous enzymes, particularly in neuronal tissues, with the ability to treat multigenetic diseases and neuronal disorders.
Implementation Method 1
The H and I segments are designed to pair with the target mRNA and place the target base to be edited in an A:C mismatch
Implementation Method 2
The nucleoside adenosine can be enzymatically deaminated. This produces inosine, which is read during translation like guanosine
Data Source
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AI summary
The invention relates to methods and substances for the targeted alteration of genetic information on an RNA level. The substances are artificially produced guide RNAs, which are capable of recruiting endogenous editing enzymes, such as hADAR enzymes, in particular hADAR2 and hADAR1, in order to introduce targeted point mutations in selected mRNAs. The guide RNA consists of multiple segments and is constructed in such a way that individual nucleotides from different segments pair to form a double helix, and the nucleotides of a determined segment form a hairpin structure within the guide RNA. The invention also relates to the method for directed RNA editing, wherein the guide RNA is transfected into the cells in which the RNA editing is to be carried out. The substances and the method can be used for repairing individual, e.g. disease-relevant point mutations, such as those leading to premature stop signals. An advantage of the invention is that endogenous editing enzymes are also used in order to introduce targeted point mutations into the RNA. Only the short guide RNA, used for recruiting endogenous editing enzymes, must be artificially produced for each specific problem and ectopically expressed.