Editing Oligonucleotides with Phosphonoacetate Linkages for RNA Editing
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Solution Overview
Problem
Current methods for RNA editing, such as those using ADAR enzymes, face challenges in specificity and efficiency, particularly in therapeutic applications, as they often require genetic modification of cells and delivery of recombinant proteins, leading to promiscuous editing and limited applicability in mammals.
Innovation Solution
Development of editing oligonucleotides (EONs) that form a double-stranded complex with target nucleic acids, recruiting endogenous ADAR enzymes using phosphonoacetate internucleotide linkages and unlocked nucleic acid (UNA) ribose modifications to specifically deaminate target adenosines, avoiding positions that would disrupt enzyme interaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional RNA editing methods using ADAR enzymes are employed, then RNA editing can be achieved, but genetic modification of cells and delivery of recombinant proteins are required, increasing device complexity and difficulty of operation
Solution Approach 1:
The invention extracts and utilizes only the essential catalytic domain of ADAR enzymes, separating it from the complex natural enzyme structure. This allows the catalytic function to be retained while eliminating the need for full recombinant protein delivery and genetic modification of cells, thereby reducing device complexity while maintaining editing reliability
Solution Approach 2:
The invention introduces chemically modified oligonucleotides as intermediaries that bridge the gap between simple chemical compounds and complex biological enzymes. These oligonucleotides with specific chemical modifications (phosphonoacetate linkages, UNA ribose modifications) serve as mediators that recruit endogenous ADAR enzymes to target sites without requiring genetic modification or recombinant protein delivery
2Productivity
If conventional RNA editing methods are used, then editing can occur, but promiscuous editing and off-target effects increase, decreasing manufacturing precision
Solution Approach 1:
The invention applies local quality by introducing specific chemical modifications at precise positions within the oligonucleotide structure. Phosphonoacetate linkages and UNA ribose modifications are placed at specific locations to create localized structural features that enhance target recognition and ADAR recruitment, thereby improving editing specificity without sacrificing productivity
Solution Approach 2:
The invention changes physical and chemical parameters of the oligonucleotide structure, including backbone chemistry (phosphonoacetate linkages), sugar modifications (UNA ribose), and secondary structure formation. These parameter changes create oligonucleotides with optimized properties for specific ADAR recruitment and target recognition, reducing off-target effects while maintaining high editing throughput
3Reliability
If chemically modified oligonucleotides with specific structures are designed to recruit ADAR enzymes, then RNA editing specificity and efficiency are enhanced, but oligonucleotide structure complexity increases
Solution Approach 1:
The invention segments the oligonucleotide structure into distinct functional regions: phosphonoacetate linkage regions for structural stability and ADAR interaction, UNA ribose modification regions for enhanced binding properties, and sequence regions for target recognition. This segmentation allows each component to contribute specifically to editing efficiency without requiring complete structural redesign
Solution Approach 2:
The invention creates composite oligonucleotide structures combining different chemical components (phosphonoacetate linkages, UNA ribose modifications, standard nucleotides) in a single molecule. This composite approach leverages the advantageous properties of each component to achieve high editing efficiency while managing overall structural complexity through rational design
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
The EONs enhance RNA editing specificity and efficiency by utilizing natural ADAR enzymes within cells, reducing off-target effects and enabling therapeutic RNA editing in mammalian cells without the need for genetic modification or recombinant protein delivery.
Implementation Method 1
capable of recruiting an enzyme with nucleotide deaminase activity, wherein the target nucleic acid molecule comprises a target nucleotide for deamination by the enzyme with nucleotide deamination activity
Implementation Method 2
capable of forming a double stranded complex with a target nucleic acid molecule in a cell
Data Source
AI summary
The invention relates to editing oligonucleotides (EONs) for binding to a target nucleic acid and recruiting an enzyme with nucleotide deamination activity to edit the target nucleic acid. The EONs carry phosphonoacetate internucleotide linkage modifications and/or unlocked nucleic acid (UNA) ribose modifications at specified positions and do not carry such modifications on positions that would lower nucleic acid editing efficiency. The selection of positions that should or should not carry a modification is based on computational modelling that revealed incompatibilities of the modifications with the enzyme with nucleotide deamination activity.