Linear Plasmid System for Stable Long Poly(A) Tracts
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Solution Overview
Problem
Current methods for large-scale generation of in vitro transcribed (IVT) mRNA are limited by the instability of homolypolymeric tracts in circular plasmids, which restricts the length of poly(A) tails and thereby limits the efficiency of translation.
Innovation Solution
The use of a linear plasmid system that can stably propagate poly(A) tracts up to 500 base pairs in length, along with modifications such as removing extraneous restriction sites, adding a T7 promoter sequence, and incorporating a unique Type IIS restriction site, to facilitate the generation of IVT mRNA with precisely defined poly(A) tracts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If circular plasmid templates are used for large scale IVT mRNA generation, then the plasmid system is stable and easy to propagate, but the homolypolymeric tracts (poly(A) tails) become highly unstable and limit encoded poly(A) tail lengths to less than approximately 120 base pairs
Solution Approach 1:
The patent divides the plasmid into linear segments with defined ends, separating the poly(A) tract from the circular backbone. This segmentation allows the poly(A) tract to be propagated as a stable linear element rather than being constrained by circular plasmid topology, resolving the contradiction between plasmid stability and poly(A) tail length.
Solution Approach 2:
The patent inverts the conventional circular plasmid approach by using a linear plasmid backbone. This inversion transforms the unstable homolypolymeric tracts in circular plasmids into stable linear poly(A) tracts that can be propagated without the topological constraints of circular DNA, enabling lengths greater than 120 base pairs.
2Length of moving object
If linear plasmid system is used to propagate poly(A) tracts, then poly(A) tail lengths up to 500 base pairs can be achieved, but the plasmid system becomes more complex requiring modifications such as removing restriction sites and adding promoters
Solution Approach 1:
The patent extracts and removes extraneous restriction sites from the linear plasmid backbone, retaining only the essential elements for poly(A) tract propagation. This extraction simplifies the plasmid system by eliminating unnecessary components while maintaining the capability to generate long poly(A) tracts.
Solution Approach 2:
The patent creates a universal linear plasmid system with a T7 promoter and unique Type IIS restriction site that can be applied to generate various poly(A) tail lengths and mRNA products. This multi-functional design simplifies the overall system by providing a single platform for multiple applications.
3Productivity
If homolypolymeric tracts are used in circular plasmids, then the plasmid can be propagated, but the tracts become highly unstable and limit translation efficiency
Solution Approach 1:
The patent inverts the conventional circular plasmid approach by using a linear plasmid backbone. This inversion transforms the unstable homolypolymeric tracts in circular plasmids into stable linear poly(A) tracts that can be propagated without the topological constraints of circular DNA, resolving the contradiction between plasmid stability and poly(A) tail length.
Solution Approach 2:
The patent changes the topological parameter of the plasmid from circular to linear, fundamentally altering the stability characteristics of the poly(A) tracts. This parameter change enables the tracts to maintain stability over longer lengths, directly improving translation efficiency.
Data Source
Figure 1A~1B
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AI summary
Disclosed herein are polynucleotides having a plurality of thymine nucleotides and an endonuclease recognition site inserted therein, methods of engineering the polynucleotides having a plurality of thymine nucleotides and an endonuclease recognition site inserted therein, and methods of enhancing transcription, translation, and increasing stability of a polynucleotide.