Aptamer Riboswitch Splicing Control for Timed Gene Expression
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Solution Overview
Problem
Existing technologies lack effective methods for regulating the expression of target genes, particularly in therapeutic contexts, where controlled and timed expression is crucial for safety and efficacy.
Innovation Solution
A polynucleotide cassette comprising a riboswitch and an alternatively-spliced exon, flanked by introns, which utilizes an aptamer to bind small molecule ligands, controlling splicing and gene expression through a riboswitch effector region that forms a stem to prevent or allow inclusion of the exon based on ligand presence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods are used for gene expression regulation, then the system is simple to implement, but the expression control precision and inducibility are insufficient
Solution Approach 1:
The patent introduces an aptamer as an intermediary component that specifically binds to small molecule ligands, translating chemical signals into conformational changes in the riboswitch. This aptamer-mediated mechanism enables precise control of alternative splicing events, achieving high inducibility (up to 1687-fold expression induction) while maintaining modular system architecture that can be integrated into existing gene expression frameworks
2Reliability
If no regulation system is used, then the gene expression is constitutive and simple, but the safety and timing control are inadequate for therapeutic applications
Solution Approach 1:
The patent implements a dynamic regulation system where the riboswitch can switch between two stable conformational states (induced and uninduced) based on ligand presence. The alternative exon inclusion/exclusion mechanism provides binary on/off control of gene expression, enabling temporal and conditional regulation that enhances therapeutic safety by allowing expression only when and where needed, while the modular cassette design minimizes the complexity burden
3Productivity
If the riboswitch stem structure is stabilized, then the alternatively-spliced exon is excluded and gene expression is induced, but the baseline expression control becomes challenging
Solution Approach 1:
The patent employs parameter optimization of the riboswitch stem structure, adjusting stem length, GC content, and sequence composition to achieve the desired balance between induced and baseline expression levels. By tuning these structural parameters, the system achieves high inducibility (up to 1687-fold) while maintaining controllable baseline expression, demonstrating that parameter optimization can resolve the trade-off between expression induction and baseline control
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 system achieves up to 1687-fold induction of target gene expression in response to ligands, providing precise control over gene expression levels and timing.
Implementation Method 1
The riboswitch comprises (i) an effector region comprising a stem that includes the 5′ splice site of the 3′ intron, and (ii) an aptamer, wherein the alternatively-spliced exon comprises a stop codon that is in-frame with the target gene when the alternatively-spliced exon is spliced into the target gene mRNA. In one embodiment, the aptamer specifically binds a small molecule ligand.
Data Source
AI summary
The invention provides a platform and methods of using the platform for the regulation of the expression of a target gene using exposure to an aptamer ligand (for example, a small molecule). The platform features a polynucleotide gene regulation cassette that is placed in the target gene and includes a synthetic riboswitch positioned in the context of a 5′ intron-alternative exon-3′ intron. The riboswitch comprises an effector region and a sensor region (e.g., an aptamer that binds a small molecule ligand) such that the alternative exon is spliced into the target gene mRNA when the ligand is not present thereby preventing expression of the target gene. When the ligand is present, the alternative exon is not spliced into the target gene mRNA thereby providing expression of the target gene.