Circular Guide RNA Complex for Selective Stop Codon Pseudouridylation
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Solution Overview
Problem
Current nucleic acid molecules for pseudouridylation in mammalian cells lack sufficient selectivity and efficacy in targeting premature stop codons in RNA, which are responsible for genetic diseases such as cystic fibrosis and Duchenne muscular dystrophy.
Innovation Solution
A nucleic acid molecule complex composed of guide RNA (gRNA) flanked by twister ribozymes, forming a circular structure with pseudouridylation pockets, enhances targeted pseudouridylation by increasing stability and specificity, utilizing a promoter and engineered RNA editing organelles to recruit pseudouridine synthase enzymes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid molecules are used for pseudouridylation, then the basic modification function is achieved, but selectivity and efficacy in targeting premature stop codons are insufficient
Solution Approach 1:
The nucleic acid molecule is divided into distinct functional regions: a guide region complementary to the target RNA sequence, a pseudouridylation pocket containing the catalytic site, and flanking sequences. This segmentation allows each region to be optimized independently for its specific function, improving both selectivity (guide region) and efficacy (pseudouridylation pocket).
Solution Approach 2:
Different regions of the nucleic acid molecule have different structural and functional properties. The guide region is optimized for sequence complementarity and binding specificity, while the pseudouridylation pocket is structured to facilitate catalytic activity. This local differentiation of functional properties enables simultaneous improvement of selectivity and efficacy.
2Stability of the object's composition
If linear gRNA structure is used, then simplicity of construction is maintained, but stability and persistence in mammalian cells are reduced
Solution Approach 1:
The guide region and pseudouridylation pocket are merged into a single continuous nucleic acid molecule with a stable secondary structure. The flanking sequences are integrated into the molecule, creating a unified construct that maintains structural integrity and stability in mammalian cells while performing both guide and catalytic functions.
3Productivity
If pseudouridylation is applied to target RNA, then premature stop codon readthrough is achieved, but off-target effects and lack of specificity occur
Solution Approach 1:
The pseudouridylation catalytic activity is extracted and concentrated within a defined pseudouridylation pocket region, separated from the guide region. This allows the catalytic site to be optimized for efficiency while the guide region maintains high specificity through sequence complementarity, preventing off-target effects.
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 circular gRNA structure significantly improves pseudouridylation efficiency, allowing the ribosome to read through premature stop codons, producing full-length functional proteins and mitigating genetic diseases like cystic fibrosis and Duchenne muscular dystrophy.
Implementation Method 1
Pseudouridylation is the process of converting uridine (U) to pseudouridine (Ψ) in RNA molecules. This is the most abundant RNA modification and involves the isomerization of uridine, where the uracil base is attached to the ribose via a carbon-carbon (C-C) bond instead of the usual nitrogen-carbon (N-C) bond.
Implementation Method 2
twister ribozymes, which are able to form the pseudouridylation pockets in the generated circular gRNAs
Data Source
Figure 1a~1d
Figure 2a~2d
Figure 3a~3b
AI summary
The invention relates to a nucleic acid molecule complex for pseudouridylation of a target RNA in a mammalian cell comprising a guide RNA (gRNA) that at one end is flanked by a first twister ribozyme and at its other end by a second twister ribozyme to generate a circular gRNA, wherein the nucleic acid molecule complex has the capability to generate circular gRNA which is characterized by a secondary structure comprising two pseudouridylation pockets that harbor antisense guide sequences. More specifically, the first twister ribozyme is a P3 twister ribozyme and the second twister ribozyme is a P1 twister ribozyme, wherein each twister ribozyme is separated from the gRNA by a linker. In a further aspect, the invention relates to a method for generating circular gRNA. The invention furthermore relates to a nucleic acid molecule complex as defined herein for use in the treatment of a genetic disease caused by a premature stop codon. It furthermore relates to a pharmaceutical composition, and a delivery vector to express the construct in mammalian cells to generate circular gRNA.