Autocatalytic RNA Sensor for Gene Expression Modulation
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Solution Overview
Problem
Current genetic circuits face challenges in modulating gene expression effectively across different tissues due to varying levels of endogenous adenosine deaminases, leading to inefficiencies in RNA-responsive systems, particularly in tissues with low ADAR levels, where overexpression of exogenous ADAR can increase cellular resource consumption and delivery complexities.
Innovation Solution
Design of nucleic acids comprising a translation initiation sequence, a sensor sequence with a premature stop codon, and a base editor sequence, separated by self-cleaving peptides, which utilize endogenous ADAR for initial editing and amplify ADAR expression to enhance dynamic range without overloading cells, incorporating ADAR-mediated RNA-responsive sensors that can detect trigger nucleic acids and activate translation of therapeutic outputs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If exogenous ADAR is overexpressed to enhance sensor performance in tissues with low endogenous ADAR levels, then the dynamic range and effectiveness of RNA-responsive sensors are improved, but the number of transcriptional units increases and cellular resource consumption increases
Solution Approach 1:
The patent combines the ADAR enzyme coding sequence with the sensor sequence into a single transcriptional unit. The ADAR enzyme and sensor are merged into one open reading frame separated by a self-cleaving 2A peptide sequence, allowing co-expression from a single promoter and reducing the number of transcriptional units from two to one.
Solution Approach 2:
The sensor design enables self-amplification where the initial editing by endogenous or supplied ADAR creates a functional sensor that then produces additional ADAR enzyme. The system uses its own output (ADAR enzyme) to enhance its performance, creating a positive feedback loop that amplifies the sensing capability without requiring continuous external supplementation.
2Reliability
If exogenous ADAR is overexpressed to enhance sensor performance in tissues with low endogenous ADAR levels, then the dynamic range and effectiveness of RNA-responsive sensors are improved, but delivery complexities increase
Solution Approach 1:
The patent combines the ADAR enzyme coding sequence with the sensor sequence into a single transcriptional unit. The ADAR enzyme and sensor are merged into one open reading frame separated by a self-cleaving 2A peptide sequence, allowing co-expression from a single promoter and reducing the number of transcriptional units from two to one.
Solution Approach 2:
The system is designed to work with minimal initial ADAR activity. A small amount of endogenous or supplied ADAR is sufficient to initiate the editing process, after which the system self-amplifies. This preliminary action requirement reduces delivery complexity compared to systems requiring high levels of exogenous enzyme delivery.
3Productivity
If ADAR-mediated base editing is used to convert stop codons to sense codons, then RNA-responsive translational control is achieved, but the system requires specific dsRNA structures and mismatched adenosines
Solution Approach 1:
The patent introduces local variation in the sensor design by placing specific mismatched adenosine residues at defined positions within the dsRNA structure. These localized mismatches create the necessary recognition sites for ADAR enzyme while maintaining the overall dsRNA structure. The mismatches are strategically positioned to ensure proper enzyme binding and editing activity.
Solution Approach 2:
The system exploits changes in RNA structure and sequence parameters. By modifying specific nucleotide positions to create mismatches and by controlling the dsRNA structure formation, the system optimizes ADAR binding and editing efficiency. The self-cleaving 2A peptide parameter also enables protein separation while maintaining co-translational coupling.
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 enables precise and efficient gene expression modulation in various tissues by leveraging endogenous ADAR levels, reducing unnecessary ADAR overexpression and improving delivery efficiency, while maintaining high dynamic range and specificity in response to trigger nucleic acids.
Implementation Method 1
ADARs efficiently edit mismatched adenosines within imperfect double-stranded RNA (dsRNA) structures
Implementation Method 2
Transcripts of interest (or trigger nucleic acids) can be detected upon specific hybridization with RNA-responsive sensors
Implementation Method 3
separated by self-cleaving peptides
Data Source
AI summary
Genetic circuits that control transgene expression in response to pre-defined transcriptional cues would enable the development of smart therapeutics. The present disclosure relates to engineered programmable single-transcript RNA sensors in which adenosine deaminases acting on RNA (ADARs) autocatalytically convert trigger hybridization into a translational output. This system amplifies the signal from editing by endogenous ADAR through a positive feedback loop. Amplification is mediated by the expression of a hyperactive, minimal ADAR variant and its recruitment to the edit site via an orthogonal RNA targeting mechanism. This topology confers high dynamic range, low background, minimal off-target effects, and a small genetic footprint. The circuits and systems disclosed herein leverage an ability to detect single nucleotide polymorphisms and modulate translation in response to endogenous transcript levels in mammalian cells.