Modified Poly-A Sequence Structure for Stable Protein Expression
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Solution Overview
Problem
Existing poly A sequences in DNA constructs shorten rapidly in bacteria, leading to reduced stability and efficiency in protein expression, posing challenges in genetic engineering and genetic vaccines.
Innovation Solution
A polynucleotide encoding a modified poly-adenyl sequence with adenine to non-adenine (non-A) bases from 3:1 to 30:1, concretely 5:1, and more concretely 8:1 to 20:1, and more concretely 8:1, are used to produce the target protein, not only its length is maintained for a long period of time within a biological sample, such as E. coli, but also the expression efficiency of the target protein by the mRNA resulting from its transcription is dramatically improved.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If conventional poly A sequences (consecutive dA:dT base pairs) are used in DNA constructs, then the sequence is simple and easy to synthesize, but the sequence shortens rapidly in bacteria leading to reduced stability
Solution Approach 1:
The patent modifies the poly A sequence by introducing non-adenine bases (such as guanine, cytosine, or thymine) at specific positions within the poly A tail. This changes the compositional parameters of the sequence from 100% adenine to a mixture containing non-adenine bases, which prevents rapid shortening in bacteria while maintaining synthesis feasibility. The modified sequence maintains stability over extended periods in biological samples.
2Stability of the object's composition
If the poly A sequence length is extended to maintain stability, then the sequence stability improves, but the DNA construct becomes more complex and difficult to manipulate
Solution Approach 1:
The patent applies local quality modification by introducing non-adenine bases at specific locations within the poly A tail rather than uniformly throughout. The modified bases are strategically positioned to create stability-enhancing motifs (such as non-A-A-A-A-A-A-A-A-A-A patterns) that prevent shortening while keeping the overall sequence manageable in length and complexity.
3Productivity
If conventional poly A sequences are used, then the DNA construct is simple to produce, but the protein expression efficiency is reduced due to rapid sequence shortening
Solution Approach 1:
The modified poly A sequence with non-adenine bases introduced at specific positions maintains continuous stability over extended periods, preventing the rapid shortening that occurs with conventional sequences. This continuity of structural integrity ensures sustained protein expression efficiency throughout the lifespan of the DNA construct in bacterial cells.
Data Source
AI summary
The present invention relates to a modified poly A sequences and DNA sequences encoding the same that maintain its structural stability over an extended period within a biological sample. The modified poly-A sequence and the DNA sequences encoding the same according to the present invention possess an optimal full-length and regularly incorporate non-adenine (non-A) bases at appropriate positions, whereby the sequences are barely apt to decrease in length even within bacteria, ensuring a robust biological function as genetic material. Furthermore, the present invention may be beneficially utilized to stably produce a therapeutically effective amount of a target protein through an optimal poly A tail structure that enables the most efficient protein expression both in vivo and in vitro.