Engineered ADAR Guide RNA Scaffolds for Efficient Base Editing
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Solution Overview
Problem
Current RNA editing technologies face challenges in efficiently recruiting ADAR proteins to target RNA sequences, leading to suboptimal editing efficiency and specificity, particularly in treating genetic disorders.
Innovation Solution
Development of vectors and nucleic acids with engineered RNA editing entity recruiting domains, such as Alu and APOBEC domains, that enhance the recruitment of ADAR proteins to specific RNA targets, reducing the formation of secondary structures and improving editing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional RNA editing technologies are used, then the system is simple to implement, but the recruitment efficiency of ADAR proteins to target RNA sequences is low
Solution Approach 1:
The patent introduces intermediary recruiting domains (such as Alu domains or APOBEC binding sites) that act as mediators between the guide RNA and ADAR proteins. These domains are incorporated into the RNA editing system to enhance ADAR recruitment efficiency without requiring complete redesign of the core editing machinery, thus balancing improved productivity with acceptable system complexity.
Solution Approach 2:
The patent creates composite RNA structures by combining guide RNA sequences with specific recruiting domains (e.g., Alu domains, APOBEC binding sites). This composite approach allows the system to leverage the targeting capability of guide RNA while incorporating specialized domains that enhance ADAR protein recruitment, thereby improving overall editing efficiency without excessive complexity.
2Reliability
If RNA sequences form secondary structures with stem-loops, then the structure provides stability, but the recruitment of ADAR proteins is hindered
Solution Approach 1:
The patent applies local quality by designing RNA structures where specific regions (recruiting domains) are kept single-stranded and accessible, while other regions may form stable secondary structures. This localized approach ensures that the recruiting domains maintain their ability to bind ADAR proteins effectively, while the overall RNA structure retains stability through controlled secondary structure formation in non-critical regions.
Solution Approach 2:
The patent introduces dynamic elements into the RNA structure design, allowing the RNA to adopt different conformations. The recruiting domains are designed to remain flexible and accessible for ADAR binding, while other parts of the RNA can form stable structures. This dynamic design enables the RNA to balance structural stability with the functional requirement of ADAR recruitment.
3Productivity
If the polynucleotide sequence has high sequence identity to Alu or APOBEC domains, then the recruitment of ADAR or APOBEC proteins is enhanced, but the specificity of targeting may be reduced
Solution Approach 1:
The patent segments the RNA structure into distinct functional modules: a guide region for target recognition and a recruiting domain for protein binding. The recruiting domain (with high identity to Alu or APOBEC sequences) is separated from the guide region, allowing it to optimize protein recruitment without compromising the specificity of target RNA binding. This segmentation enables independent optimization of each function.
Solution Approach 2:
The patent applies local quality by assigning different sequence characteristics to different regions of the RNA. The guide region maintains high specificity for the target RNA sequence, while the recruiting domain incorporates sequences with high identity to Alu or APOBEC domains to maximize protein recruitment. This regional differentiation resolves the contradiction between recruitment efficiency and targeting specificity.
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 engineered RNA editing systems demonstrate enhanced editing efficiency, enabling effective treatment of conditions like muscular dystrophy and neurodegenerative disorders by modifying gene expression and correcting point mutations.
Implementation Method 1
ADAR proteins are adenosine deaminases that act on RNA and can be recruited to a target RNA
Implementation Method 2
APOBEC proteins are cytidine deaminases that act on RNA and can be recruited to a target RNA
Data Source
AI summary
Disclosed herein is a system to recruit ADARs to catalyze therapeutic editing of point mutations via the use of engineered RNA scaffolds, engineered DNA scaffolds or DNA-RNA hybrid scaffolds. The system comprises an engineered ADAR2 guide RNA (adRNA) that bears a 20-100 bp complementarity with the target RNA and ADAR2 recruiting domain from the GluR2 mRNA at either or both 5′ end or 3′ end.