Artificial Riboswitch for Conditional Pre-mRNA Splicing Control
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Solution Overview
Problem
Current methods for controlling pre-mRNA splicing are limited by their constitutive nature, susceptibility to nuclease digestion, off-target effects, and difficulties in tissue-specific regulation, necessitating the development of novel approaches for condition-specific modulation of RNA splicing.
Innovation Solution
The use of artificial riboswitches, specifically designed to regulate pre-mRNA splicing in response to theophylline binding, which modulates the splicing process by altering the binding affinity and location of splicing sites within the RNA sequence, allowing for conditional control of splicing events.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional splicing control methods are used, then splicing regulation is achieved, but the methods are constitutive and lack condition-specific control
Solution Approach 1:
The patent introduces dynamic control of splicing through riboswitches that can change conformation in response to ligand binding. The riboswitch structure transitions between different states (with or without ligand bound), dynamically controlling splice site accessibility and splicing outcomes based on cellular conditions.
Solution Approach 2:
The riboswitch acts as an intermediary molecule between the splicing machinery and external signals. It receives ligand binding signals and translates them into structural changes that modulate splicing factor binding and splice site selection, providing condition-specific control without directly interacting with the complex splicing machinery.
2Reliability
If antisense oligonucleotides are used for splicing control, then splicing modulation is achieved, but susceptibility to nuclease digestion and off-target effects occur
Solution Approach 1:
The riboswitch is an endogenous RNA element that is transcribed as part of the target pre-mRNA itself. It regulates splicing through its own structural properties and ligand binding capabilities, eliminating the need for externally introduced oligonucleotides that are susceptible to nucleases and off-target effects.
Solution Approach 2:
The riboswitch regulatory sequence is merged with the pre-mRNA transcript, creating a unified molecule where the regulatory element and the target sequence are covalently linked. This ensures that the riboswitch only regulates its own transcript, preventing off-target effects and eliminating the need for separate therapeutic oligonucleotide administration.
3Ease of operation
If tissue-specific regulation is implemented, then precise spatial control is achieved, but delivery and localization difficulties arise
Solution Approach 1:
The riboswitch confers local regulatory capability directly at the site of gene transcription within each cell type. Tissue-specific control is achieved through the intrinsic properties of the riboswitch and the cellular distribution of its ligand, eliminating the need for complex delivery systems to target specific tissues.
Solution Approach 2:
Each cell type autonomously regulates its own splicing through the riboswitch present in its transcripts. The riboswitch responds to locally available ligands (such as metabolites that differ by tissue type), enabling self-regulated tissue-specific control without external intervention or complex delivery mechanisms.
Data Source
AI summary
The present invention relates to riboswitches that have been engineered to regulate pre-mRNA splicing. In particular, the insertion of a high affinity theophylline binding aptamer into the 3′ splice site region, 5′ splice site region, or branchpoint sequence (BPS) of a pre-mRNA modulates RNA splicing in the presence of theophylline. Accordingly, the aspects of the present invention include, but are not limited to, theophylline-dependent riboswitches which modulate RNA splicing, methods of modulating RNA splicing using theophylline and its corresponding riboswitches, methods of improving/identifying theophylline-dependent riboswitches, methods of treating diseases associated with or caused by abnormal RNA splicing.