Codons 3–5 Sequence Tuning for Precise Protein Expression

Solution Overview

Problem

Existing methods for manipulating gene expression are time-consuming, organism-specific, costly, and lack the ability to tightly control protein levels, making them inconsistent and inefficient.

Innovation Solution

Modifying the nucleic acid sequence at codons 3, 4, and 5 by inserting or changing nucleotides to create a modified nucleic acid sequence that encodes a second protein, which can be expressed in a cell or cell-free system, allowing for precise control over protein expression levels.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If existing methods for manipulating gene expression are used (manipulating gene copy numbers, using RNAi, varying promoters, optimizing coding sequences), then protein levels can be altered, but the process is time-consuming, costly, and lacks tight control over protein expression levels

Engineering Contradiction:

Improvecontrol precision of protein expression levelsVSAvoidtime required for gene expression manipulation

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The invention changes the nucleotide parameters at specific codon positions (3, 4, and 5) to modulate translation efficiency. By systematically varying the nucleotide sequences at these specific positions while keeping the amino acid sequence constant, the method achieves precise control over protein expression levels without requiring time-consuming manipulations of gene copy numbers, promoters, or extensive coding sequence optimization

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention applies local quality by focusing modifications only on specific codon positions (3, 4, and 5) rather than globally optimizing the entire coding sequence. This localized approach to codon optimization allows for rapid testing and tuning of expression levels by making targeted nucleotide changes at these critical positions, significantly reducing the time and resources required compared to comprehensive gene optimization

Inventive Principle:

Principle #3Local quality

2Measurement precision

If existing methods for manipulating gene expression are used, then protein levels can be altered, but the approach is costly and inconsistent

Engineering Contradiction:

Improvecontrol precision of protein expression levelsVSAvoidcost and complexity of gene expression manipulation

Core Design Contradiction:

Measurement precisionVSEase of manufacture

Solution Approach 1:

The invention changes the nucleotide parameters at specific codon positions (3, 4, and 5) to modulate translation efficiency. By systematically varying the nucleotide sequences at these specific positions while keeping the amino acid sequence constant, the method achieves precise control over protein expression levels without requiring time-consuming manipulations of gene copy numbers, promoters, or extensive coding sequence optimization

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention segments the coding sequence into specific codon positions (3, 4, and 5) that have disproportionate impact on translation efficiency. By isolating and optimizing only these critical positions rather than the entire gene sequence, the method reduces the complexity and cost of manufacturing while maintaining precise control over expression levels

Inventive Principle:

Principle #1Segmentation

3Measurement precision

If existing methods for manipulating gene expression are used, then protein levels can be altered, but the ability to tightly control the extent of protein level changes is limited

Engineering Contradiction:

Improvecontrol precision of protein expression levelsVSAvoidcomplexity of gene expression manipulation system

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The invention changes the nucleotide parameters at specific codon positions (3, 4, and 5) to modulate translation efficiency. By systematically varying the nucleotide sequences at these specific positions while keeping the amino acid sequence constant, the method achieves precise control over protein expression levels without requiring time-consuming manipulations of gene copy numbers, promoters, or extensive coding sequence optimization

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention extracts the critical control elements for translation efficiency into specific codon positions (3, 4, and 5), separating them from the rest of the coding sequence. This extraction allows for independent optimization of these positions to achieve desired expression levels without complicating the overall gene structure or requiring complex regulatory elements

Inventive Principle:

Principle #2Taking out (Extraction)

Data Source

PatentUS12428640B2Methods to modulate protein translation efficiency

Publication Date: 2025.09.30 WASHINGTON UNIV IN SAINT LOUIS

AI summary

The present disclosure relates to compositions and methods to modulate the level of expression of a protein in a deliberate manner (i.e., tunable regulation of expression) with only a minimal change to the genetic sequence of the gene of interest. The present disclosure therefore also provides compositions and methods to predictably alter protein abundance.