Engineered Guide RNA Structural Features for SNCA Editing
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Solution Overview
Problem
Current RNA editing compositions face challenges in maximizing on-target RNA editing while minimizing off-target editing, particularly for genetic diseases like Parkinson's, where precise editing of SNCA RNA is needed to reduce alpha-synuclein protein aggregation.
Innovation Solution
Engineered guide RNAs that form specific structural features upon hybridization with SNCA RNA, such as bulges, internal loops, and hairpins, to facilitate targeted RNA editing by ADAR enzymes, enhancing specificity and reducing off-target edits.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional guide RNAs are used for RNA editing, then the editing process can be initiated, but off-target editing occurs and editing precision is insufficient
Solution Approach 1:
The guide RNA is engineered with position-specific structural features (bulges, internal loops, hairpins) at defined locations relative to the target adenosine. These localized structural modifications create distinct binding characteristics that enhance specificity for the target site while minimizing off-target effects, directly resolving the precision versus off-target editing contradiction
Solution Approach 2:
The guide RNA incorporates asymmetric structural features including 6/6 symmetric internal loops at specific positions (e.g., position 32, 30, 28, 26, or 24 relative to the target adenosine) combined with asymmetric bulges and mismatches. This asymmetric design creates a unique binding profile that distinguishes the target site from similar sequences, thereby improving editing precision and reducing off-target editing
2Reliability
If guide RNAs with high complementarity to target RNA are used, then on-target binding is strengthened, but structural complexity increases
Solution Approach 1:
The guide RNA is divided into functional segments with specific structural features positioned at defined distances from the target adenosine. The 6/6 symmetric internal loop is positioned at specific positions (e.g., 32, 30, 28, 26, or 24 nucleotides relative to the target adenosine), with additional features like bulges and mismatches at specific locations. This segmentation allows each feature to contribute independently to binding reliability while maintaining manageable structural complexity
Solution Approach 2:
The guide RNA design systematically varies structural parameters including the position, size, and type of structural features (bulges, internal loops, hairpins, mismatches). By optimizing these parameters—such as positioning the 6/6 symmetric internal loop at specific distances and incorporating specific mismatch types (A/C mismatch, U/C mismatch, G/G mismatch)—the design achieves reliable on-target binding without excessive structural complexity
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 guide RNAs achieve high on-target editing efficiency with reduced off-target effects, effectively knocking down alpha-synuclein protein levels, thereby treating Parkinson's disease symptoms and reducing protein aggregation.
Implementation Method 1
the engineered guide RNA, upon hybridization to a sequence of a target SNCA RNA, forms a guide-target RNA scaffold with the sequence of the target SNCA RNA
Implementation Method 2
the engineered guide RNA facilitates RNA editing of one or more target adenosines in the sequence of the target SNCA RNA by an RNA editing entity
Data Source
AI summary
Disclosed herein are engineered guide RNAs and compositions comprising the same for treatment of diseases or conditions in a subject. Also disclosed herein are methods of treating diseases or conditions in a subject by administering engineered guide RNAs or pharmaceutical compositions described herein.