Segmented PolyA Polynucleotide for Stable mRNA Plasmid Fermentation
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Solution Overview
Problem
Current methods for producing mRNA vaccines and medicaments face challenges in controlling the length of PolyA tails during transcription, leading to uncontrollable sequence changes due to recombination in host cells, which affects the efficacy of mRNA products.
Innovation Solution
A multi-segmented PolyA polynucleotide is introduced, where adjacent PolyA sequence segments are connected via linkers, stabilizing the sequence in host cells like Escherichia coli, reducing recombination and ensuring consistent PolyA tail length.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If a continuous PolyA sequence is constructed onto a plasmid for mRNA production, then the PolyA tail can be added during transcription, but the plasmid is prone to recombination during replication in host cells, resulting in uncontrollable changes in the PolyA sequence
Solution Approach 1:
The continuous PolyA sequence is divided into multiple segments separated by linker sequences. This segmentation prevents homologous recombination between adjacent PolyA repeats while maintaining the functional PolyA tail structure for mRNA transcription. The linkers act as barriers to recombination events that would otherwise occur in continuous PolyA sequences during plasmid replication in E. coli.
2Stability of the object's composition
If the PolyA sequence is modified or special strains are used to reduce recombination, then the PolyA sequence stability is improved, but the plasmid construction difficulty increases
Solution Approach 1:
The PolyA sequence is segmented into multiple units with linkers, which inherently reduces recombination without requiring special strain engineering or complex modifications. This approach maintains simplicity in plasmid construction while achieving stability.
3Length of moving object
If a long continuous PolyA sequence is used to ensure sufficient tail length, then the mRNA can exert physiological functions, but recombination events increase, leading to sequence changes and loss of control over PolyA tail length
Solution Approach 1:
The long PolyA tail is constructed by segmenting it into multiple shorter units separated by linkers. This maintains the total length required for physiological function while preventing recombination between adjacent PolyA repeats, thereby ensuring sequence consistency and reliable control over the final PolyA tail length in the mRNA product.
Solution Approach 2:
Linker sequences act as intermediaries between PolyA segments, preventing direct interaction and recombination between PolyA repeats while allowing the segments to function together as a complete PolyA tail during transcription.
4Ease of manufacture
If the PCR method is used to add PolyA to the transcription template, then the PolyA tail can be added, but the method is limited to small-scale production due to PCR system limitations
Solution Approach 1:
The PolyA sequence is pre-constructioned into the plasmid template in a segmented format, eliminating the need for subsequent PCR-based PolyA addition. This preliminary incorporation enables large-scale plasmid replication and mRNA production through transcription, overcoming the productivity limitations of PCR-based methods.
Data Source
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AI summary
A multi-segmented PolyA polynucleotide stably existing in a host cell and the use thereof. Provided first is a multi-segmented PolyA polynucleotide comprising no less than two PolyA sequence segments. Adjacent PolyA sequence segments are connected via a linker, wherein each PolyA sequence segment is composed of a plurality of continuous A, and each linker is composed of 1-24 nt nucleotide residues that are not all A. The multi-segmented PolyA polynucleotide can stably exist in a host cell, such as an Escherichia coli, during the passage process, so that the mRNA stability and protein output can be improved. Compared with a poly A sequence in a traditional unit form, the multi-segmented PolyA polynucleotide shows a greatly reduced recombination rate during fermentation in Escherichia coli and thereby is more advantageous for optimization of the amplification process, and therefore can be better applied to plasmid fermentation involved in transcription template preparation in production of mRNA vaccines or medicaments.