Protein Production Through Optimized Orthogonal mRNA Binding
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Solution Overview
Problem
Existing methods for orthogonal protein production using orthogonal ribosomes and tRNA pairs result in low protein yields, as the current O-ribosome binding sites do not provide a general solution for efficient translation of open reading frames.
Innovation Solution
A method is developed to optimize orthogonal messenger RNA (O-mRNA) sequences by predicting and modifying the 5' untranslated region (UTR) to enhance binding with orthogonal ribosomes, using thermodynamic calculations and simulated annealing to ensure efficient protein production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If orthogonal ribosomes and tRNA pairs are used for protein production, then non-canonical amino acids can be incorporated into proteins, but protein yield is low
Solution Approach 1:
The patent optimizes the 5' UTR sequence parameters of O-mRNA to improve ribosome binding. By calculating and modifying thermodynamic parameters (Delta G values) of the 5' UTR, the system achieves stronger binding between orthogonal ribosomes and mRNA, thereby increasing protein yield while maintaining the ability to incorporate non-canonical amino acids
Solution Approach 2:
The patent performs preliminary optimization of the 5' UTR sequence before translation occurs. Through computational methods, the 5' UTR is designed and modified in advance to ensure optimal ribosome binding and translation efficiency, preventing the low yield issue from arising in the first place
2Productivity
If the O-ribosome binding site is modified to improve binding efficiency, then protein production increases, but the complexity of mRNA design increases
Solution Approach 1:
The patent replaces manual trial-and-error mRNA design with computational thermodynamic calculations. By using algorithms to calculate Delta G values and predict ribosome binding, the system automates the optimization process, reducing the complexity burden on researchers while maximizing protein production
Solution Approach 2:
The optimization system is self-service in nature, where the computational tools automatically generate and evaluate 5' UTR sequences without requiring extensive manual intervention. The system self-optimizes the mRNA design based on thermodynamic principles, reducing the complexity burden on the user
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 optimized O-mRNA sequences significantly improve protein yield, achieving up to 31-fold higher production compared to unoptimized systems, enabling efficient incorporation of multiple non-canonical amino acids into proteins.
Implementation Method 1
The free energy released on forming the initiation complex from unfolded mRNA with a wt and orthogonal 30S are ΔGwt ribo binding and ΔGO-ribo binding respectively
Implementation Method 2
predicting the free energy difference between the free-folded state of the mRNA and the O-ribosome-bound initiation-competent state of the mRNA (ΔGtot(O-ribo))
Data Source
AI summary
The present invention relates to novel methods of optimising protein production. These methods include: methods of optimising orthogonal mRNAs, methods of designing and producing optimal operons comprising exogenous tRNAs, and methods of designing and producing optimal operons comprising exogenous genes, such as those encoding orthogonal aminoacyl-tRNA synthetases (O-aaRSs). The invention also relates to the products of said methods. Also provided as a part of the invention are host cells comprising the products of these innovations, methods of using said cells, and the products thereof. The host cells of the invention may be used for improved production of proteins and polypeptides comprising genetically incorporated non-canonical amino acids.