Ribozyme T-Box Flexizyme Segmentation for Unnatural Amino Acid Diversity
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Solution Overview
Problem
Current methods for introducing unnatural amino acids (uAAs) into eukaryotic cells are inefficient due to the complexity of orthogonal tRNA synthetases recognizing host cell components, limiting the chemical diversity of uAAs that can be incorporated into proteins.
Innovation Solution
Development of artificial ribozymes comprising a T-box element and a flexizyme, which recognize and bind specific tRNAs, allowing for the efficient charging of tRNAs with a wide variety of uAAs, including those that are not specific to a targeted amino acid, enabling their incorporation into proteins within eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional orthogonal tRNA synthetases are used to introduce unnatural amino acids into eukaryotic cells, then site-specific incorporation of uAAs is achieved, but the chemical diversity of uAAs that can be incorporated is limited due to recognition of host cell components
Solution Approach 1:
The invention divides the aminoacyl-tRNA synthetase function into two separate components: a tRNA recognition element (derived from the anticodon-binding domain) and a catalytic element (the aminoacylation domain). These segmented components can function independently or in combination, allowing the catalytic element to be exchanged for different amino acid specificities without redesigning the entire synthetase system. This segmentation enables greater chemical diversity of uAAs to be incorporated while reducing the complexity of designing new orthogonal pairs.
Solution Approach 2:
The catalytic element of the aminoacyl-tRNA synthetase is designed to be universal, capable of aminoacylating multiple different tRNA species with different amino acid specificities. This multi-functional catalytic domain can be paired with different tRNA recognition elements to create various orthogonal pairs for incorporating different unnatural amino acids, thereby increasing the versatility and chemical diversity of uAAs that can be introduced into eukaryotic cells without requiring entirely new synthetase systems for each application.
2Productivity
If orthogonal tRNA synthetases are engineered to recognize specific tRNAs and uAAs, then site-specific uAA incorporation is enabled, but the system becomes complex and less efficient in eukaryotic cells
Solution Approach 1:
By segmenting the aminoacyl-tRNA synthetase into separate tRNA recognition and catalytic elements, the invention simplifies the overall system architecture. The catalytic element can be optimized for high efficiency in aminoacylation reactions independently from the tRNA recognition specificity, which is handled by the separate recognition element. This segmentation allows each component to be optimized for its specific function, improving overall productivity while reducing the complexity of engineering complete orthogonal synthetase systems.
Solution Approach 2:
The invention introduces a modular interface between the tRNA recognition element and the catalytic element that acts as an intermediary. This interface allows the two components to associate and function together while maintaining their individual optimizations. The intermediary connection enables the system to achieve high productivity through efficient aminoacylation while keeping the overall system complexity manageable through modular, interchangeable components rather than requiring entirely new complex synthetase systems for each uAA application.
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 artificial ribozymes provide a robust mechanism for selectively charging tRNAs with uAAs, enhancing the chemical diversity of amino acids that can be incorporated into proteins and improving the efficiency of protein engineering in eukaryotic cells.
Implementation Method 1
The flexizyme comprises an active site for binding an amino acid and charging a tRNA molecule
Data Source
AI summary
Ribozymes exhibiting tRNA synthetase activity and substrate specificity, as well as methods for engineering and producing the same, are disclosed. The ribozymes of the present disclosure comprise a T-box module fused with a flexizyme module. The flexizyme module provides high promiscuity with respect to amino acid substrates and the T-box module provides tRNA substrate specificity, which may be engineered as desired. Systems are also described for aminoacylation of suppressor tRNAs with unnatural amino acids (uAAs), such systems comprising the ribozyme previously mentioned, suppressor tRNA, and the desired uAA(s). Methods for incorporating a uAA into a growing polypeptide chain using the ribozyme hereof are also provided.