Modifying Nucleic Acid Sequences to Reduce RNase L Cleavage Sites
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Solution Overview
Problem
Mammalian expression systems face challenges in achieving high protein expression yields, particularly for difficult-to-express proteins like membrane proteins, antibodies, and large proteins, due to inherent instability and low mRNA stability, which is exacerbated by RNase L cleavage sites in nucleic acid sequences.
Innovation Solution
Reducing the number of RNase L cleavage sites, specifically UU and UA dinucleotides, in the coding and non-coding regions of nucleic acid sequences to enhance mRNA stability and protein expression without altering the amino acid sequence or disrupting regulatory elements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If nucleic acid sequences are used for protein expression in mammalian systems, then protein production is achieved, but mRNA stability is reduced due to RNase L cleavage sites
Solution Approach 1:
The patent applies parameter changes by modifying nucleic acid sequences to alter the frequency of specific dinucleotides (UU and UA) that serve as RNase L cleavage sites. By changing the sequence composition while maintaining the encoded protein, the mRNA becomes resistant to RNase L degradation, thereby improving both mRNA stability and protein expression yield simultaneously
Solution Approach 2:
The patent converts the harmful effect of RNase L cleavage sites into a benefit by deliberately removing or reducing these sites in the nucleic acid sequence. This eliminates the degradation pathway that would otherwise reduce mRNA stability, transforming a vulnerability into a strengthened expression system with enhanced mRNA half-life and increased protein production
2Productivity
If standard nucleic acid sequences are used, then protein expression occurs, but expression yields are low for difficult-to-express proteins
Solution Approach 1:
The patent systematically modifies nucleic acid sequence parameters including dinucleotide frequency, codon usage, and sequence composition to optimize expression. These parameter changes create more reliable and consistent high-level expression across different protein types, particularly for difficult-to-express proteins like membrane proteins and antibodies
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 significantly increases protein expression yields by making mRNA more resistant to RNase L degradation, leading to improved stability and higher production levels of proteins, including therapeutic and reporter proteins, in eukaryotic cells.
Implementation Method 1
RNase L is activated by subnanomolar levels of 2-5A, resulting in the cleavage of single-stranded regions of viral RNA, preferentially after UU and UA dinucleotides in viral mRNAs
Implementation Method 2
oligoadenylate synthetase (OAS) which, upon binding to viral double-stranded RNA intermediates, becomes activated and synthesizes short 2'-5'oligoadenylates (2-5A). These, in turn, activate RNase L
Data Source
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AI summary
The present invention relates to a method for increasing the expression of a protein in cells, preferably in eukaryotic cells, by reducing the number of RNase L cleavage sites in the coding and/or non-coding region of the nucleic acid sequence of said protein. Furthermore, it relates to nucleic acid sequences exhibiting a reduced number of RNase L cleavage sites as well as to the proteins translated from such sequences.