Modulating Protein Levels via Translation Initiation Sequence SNPs
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Solution Overview
Problem
Current methods for modulating protein levels in eukaryotic organisms are challenging, particularly in non-GMO variants, as they often require extensive knowledge of the protein of interest and can result in undesired pleiotropic effects.
Innovation Solution
The method involves identifying and generating single nucleotide polymorphisms (SNPs) in the translation initiation sequence (TIS) of an endogenous gene, using a consensus matrix to determine optimal nucleotide substitutions that enhance or reduce protein levels without altering the native spatial-temporal expression profile.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional methods are used to modulate protein levels by modifying transcript level, then protein abundance can be altered, but extensive knowledge of cis-acting elements is required and GMO technology is needed
Solution Approach 1:
The invention changes the parameter of nucleotide sequence at the translation initiation site to modulate protein abundance. By altering specific nucleotides in the TIS region (positions -6 to +9 relative to AUG), the translation efficiency is changed, thereby controlling protein levels without affecting transcript stability or requiring GMO technology.
Solution Approach 2:
The invention focuses on a local region of the gene (translation initiation sequence from -6 to +9) rather than the entire gene or transcript. By concentrating the modification in this specific local area, the method achieves precise control of translation efficiency while preserving the rest of the gene's functionality and expression profile.
2Quantity of substance
If nucleotide substitutions are made to modulate protein levels, then protein abundance can be changed, but extensive knowledge of the protein of interest is required
Solution Approach 1:
The invention uses a consensus matrix that captures universal translation initiation characteristics across genes. The matrix serves as a self-contained reference that guides nucleotide substitutions without requiring extensive protein-specific knowledge. The consensus matrix itself encodes the necessary information about translation efficiency patterns.
Solution Approach 2:
The consensus matrix acts as an intermediary tool between the desired protein modulation and the actual nucleotide substitution. Instead of requiring direct knowledge of protein structure and function, the matrix provides a mediating framework that translates general translation efficiency goals into specific nucleotide changes.
3Quantity of substance
If traditional approaches are used to alter protein content, then protein levels can be modified, but undesired pleiotropic effects occur
Solution Approach 1:
By confining the nucleotide substitution to the localized translation initiation sequence (positions -6 to +9), the method affects only translation efficiency of the target gene without altering other gene expressions. This localized modification prevents pleiotropic effects that would occur with broader genetic modifications.
Solution Approach 2:
The invention segments the gene into functional regions, specifically targeting the translation initiation site as a separate controllable element. By isolating the modification to this segment, the method achieves protein level control independent of other gene functions, thereby avoiding pleiotropic consequences.
4Quantity of substance
If recombinant methods are used to modulate protein levels, then protein abundance can be changed, but the native spatial-temporal expression profile is altered
Solution Approach 1:
The method uses the gene's own translation initiation sequence as the modification target, allowing the endogenous gene to regulate its own protein levels through nucleotide substitution. This self-regulation mechanism preserves the native spatial-temporal expression pattern while achieving abundance control.
Solution Approach 2:
Instead of introducing external recombinant elements to control protein levels, the invention inverts the approach by modifying the gene's intrinsic translation initiation site. This inversion maintains the endogenous expression architecture while achieving protein abundance modulation.
Data Source
AI summary
While eliminating a protein of interest in a eukaryotic organism is relatively straightforward, non-GMO methods based on nucleotide substitutions for modulating the translational efficiency and levels of a protein towards a pre-determined aim have proven difficult. This is due to the complexity of the protein expression machinery which makes it difficult to predict and elucidate the effects of a substitution in a nucleotide sequence on the modulation of an endogenous protein expression. The present invention relates to a method for modulating levels of a protein of interest in a eukaryotic organism of a species of interest, leading to increasing the probability of either higher or lower levels of the protein of interest. Further, the present invention relates to an eukaryotic organism comprising one or more mutation(s) associated with this modulation.