Engineered Guide RNA Internal Loops for Editing Specificity
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Solution Overview
Problem
Current RNA editing technologies face challenges in achieving high specificity and efficiency for editing target adenosines in RNA molecules, often resulting in off-target editing due to the lack of precise structural features in guide RNA designs.
Innovation Solution
The development of engineered guide RNAs that form a guide-target RNA scaffold with specific structural features such as internal loops and mismatches, which enhance the editing efficiency and specificity of adenosine editing by an RNA editing entity like ADAR, by incorporating designed first and second internal loops that flank the target region.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional guide RNA designs are used for RNA editing, then the editing process can be performed, but off-target editing occurs due to lack of precise structural features
Solution Approach 1:
The patent introduces specific structural features (internal loops and mismatches) at localized positions within the guide RNA sequence. These local structural modifications create distinct binding characteristics at the target site while maintaining overall guide RNA function, thereby enhancing specificity and reducing off-target effects without requiring complete redesign of the entire guide RNA molecule.
Solution Approach 2:
The patent employs asymmetric internal loop structures with specific nucleotide compositions and lengths at defined positions relative to the target adenosine. This asymmetry creates a unique structural signature that enhances discrimination between on-target and off-target sites, as the asymmetric configuration optimizes binding affinity and editing efficiency specifically at the intended target location.
2Reliability
If guide RNA with structural features is designed to improve specificity, then off-target editing is reduced, but the design complexity increases
Solution Approach 1:
The guide RNA is divided into functional segments with specific structural elements (internal loops and mismatches) positioned at defined distances from the target adenosine. This segmentation allows systematic optimization of each element's contribution to specificity while maintaining overall design manageability through modular construction principles.
Solution Approach 2:
The patent systematically varies parameters such as internal loop length, nucleotide composition, and position relative to the target site to optimize editing specificity. By establishing quantitative relationships between these parameters and editing outcomes, the patent provides a framework for rational design that reduces complexity compared to empirical trial-and-error approaches.
3Productivity
If internal loops are added to enhance editing efficiency, then on-target editing is improved, but the structural complexity of the guide-target scaffold increases
Solution Approach 1:
The internal loop structures are pre-designed with specific configurations that facilitate optimal binding and editing complex formation. By preparing the guide RNA with these pre-optimized structural features before the editing reaction, the patent ensures high editing efficiency without requiring complex dynamic rearrangements during the reaction process, thereby managing structural complexity effectively.
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 significantly increases the editing efficiency of on-target adenosines while reducing off-target editing, as demonstrated by improved RNA editing percentages and specificity in various biological replicates, with optimal loop positions and lengths enhancing the editing efficiency.
Implementation Method 1
upon hybridization of the engineered guide RNA to a sequence of a target RNA, the engineered guide RNA and the sequence of the target RNA form a guide-target RNA scaffold
Implementation Method 2
upon contacting the guide-target RNA scaffold with an RNA editing entity, the RNA editing entity edits an on-target adenosine in the target RNA within the guide-target RNA scaffold
Data Source
AI summary
Provided herein are engineered guides configured, upon hybridization to target RNA molecules, to form double stranded RNA substrates comprising (i) a region comprising at least one structural feature; and (ii) a first internal loop and a second internal loop, wherein the double stranded RNA substrates recruit RNA editing entities and facilitate chemical modifications of base nucleotides in the target RNA molecules. Also provided herein are compositions, vectors, and cells comprising the engineered guides disclosed herein. Also provided herein are methods of introducing the engineered guides described herein into cells and methods of treating a disease or condition in a subject in need thereof comprising administering to the subject the engineered guides, polynucleotides encoding the engineered guides, delivery vehicles comprising such engineered guides or such polynucleotides, or pharmaceutical compositions comprising any one of these as described herein.