mRNA Sequence Selection for Predictable Protein Production
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Solution Overview
Problem
Current methods for protein production lack predictability due to various influencing factors such as gene copy number, location, transcription, and mRNA stability, leading to inconsistent results in heterologous protein expression.
Innovation Solution
A method involving the production of arrays of mRNA sequences encoding a polypeptide of interest, where parameters like ensemble free energy, minimum free energy RNA secondary structure, and ensemble diversity are determined and used to rank sequences, allowing for the selection of specific mRNA sequences that optimize protein production levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple factors (gene copy number, location, transcription, mRNA stability) are manipulated to increase protein production, then protein expression levels improve, but predictability and consistency of results deteriorate
Solution Approach 1:
The patent changes the parameter of codon usage in the mRNA sequence to optimize protein production. By selecting specific codons that are preferentially recognized by the host cell's tRNA pool, the invention achieves higher and more predictable translation efficiency without manipulating multiple conflicting factors simultaneously
Solution Approach 2:
The patent creates synthetic copies of the gene with optimized codon sequences. These copied and modified mRNA sequences are designed based on computational analysis of host cell codon usage patterns, allowing predictable replication of high-expression phenotypes across different production systems
2Productivity
If synthetic gene approaches with altered codon usage are used to increase protein expression, then production levels improve, but experimental variability and inconsistency increase
Solution Approach 1:
The patent employs computational feedback mechanisms where codon usage tables derived from host cell genomic data are used to guide the design of optimized mRNA sequences. This feedback loop between host cell characteristics and synthetic gene design ensures that the modified sequences are tailored to the specific production system, reducing variability
Solution Approach 2:
The invention systematically changes codon parameters based on quantitative analysis of host cell translation machinery characteristics. By adjusting codon selection according to measured tRNA abundance and codon usage bias, the patent achieves consistent, predictable expression across different experimental conditions
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 the selection of mRNA sequences that produce proteins at desired levels, enhancing or reducing production compared to wild-type levels, thereby improving the predictability and efficiency of protein expression.
Implementation Method 1
determining one or more of the following parameters for each individual mRNA sequence of (a): (i) ensemble free energy (EFE); (ii) frequency of the minimum free energy (FMFE) RNA secondary structure in a thermodynamic ensemble
Implementation Method 2
frequency of the minimum free energy (FMFE) RNA secondary structure in a thermodynamic ensemble
Data Source
AI summary
The present invention provides a method of selecting a mRNA for production of a polypeptide of interest comprising: a) producing an array of individual mRNA sequences comprising different nucleotide sequences encoding the polypeptide of interest; b) determining one or more or two or more of the following parameters for each individual mRNA sequence of (a): (i) minimum free energy (MFE) RNA secondary structure; (ii) ensemble free energy (EFE); (iii) frequency of the minimum free energy (FMFE) RNA secondary structure in a thermodynamic ensemble; and (iv) ensemble diversity (ED); c) ranking the individual mRNA sequences of the array according the parameters determined in step (b); and d) selecting a mRNA sequence from the ranked array of step (c), wherein the selected mRNA produces the polypeptide of interest. The present invention further provides a method of selecting a mRNA for enhanced and reduced production of a polypeptide of interest.