Segmented Poly(A) Tail Coding Sequence for Stable mRNA Templates
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Solution Overview
Problem
The existing methods for synthesizing mRNA drugs in vitro are unstable due to the shortening of poly (A) tail transcription template DNA in vitro, leading to reduced in vivo stability and biological activity of mRNA.
Innovation Solution
An engineered DNA molecule with a specific poly (A) tail coding sequence, comprising elements a, b, c, and d, designed to enhance stability during replication and transcription, ensuring a defined sequence for poly (A) tail stability and regulatory control.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If poly (dA:dT) repeat sequence is used in template plasmid for co-transcriptional addition of poly (A) tail, then poly (A) tail can be added to mRNA, but the sequence is unstable during replication in E. coli with deletion mutations occurring
Solution Approach 1:
The poly (A) tail coding sequence is divided into multiple segments (element a with ≥20 consecutive A nucleotides, element b with 3-19 consecutive A nucleotides, element c with non-A nucleotides, and element d with 2-20 nucleotides where terminal nucleotides are not A). These segmented elements are arranged in specific patterns to prevent deletion mutations while ensuring stable poly (A) tail production during E. coli replication.
Solution Approach 2:
Different regions of the poly (A) tail coding sequence have different nucleotide compositions and lengths optimized for specific functions. Element a provides the core poly (A) sequence, while elements b, c, and d provide local variations that prevent instability. The non-A nucleotides in element c and the specific terminal nucleotides in element d create local sequence diversity that prevents deletion mutations.
2Productivity
If poly (dA:dT) repeat sequence is used for poly (A) tail addition, then mRNA can be produced, but deletion mutations lead to shortening of poly (dA:dT) affecting in vivo stability and biological activity
Solution Approach 1:
The poly (A) tail coding sequence is pre-designed with a specific structure comprising element a (≥20 consecutive A nucleotides), element b (3-19 consecutive A nucleotides), element c (non-A nucleotides), and element d (2-20 nucleotides with non-A terminal nucleotides). This preliminary structural design prevents deletion mutations during plasmid replication, ensuring that the poly (A) tail length remains consistent throughout the mRNA production process.
Solution Approach 2:
The invention changes the parameters of the poly (A) tail coding sequence by specifying exact length ranges for different elements (element a: ≥20 nt, element b: 3-19 nt, element c: 2-20 nt, element d: 2-20 nt) and nucleotide compositions. These parameter specifications ensure that the poly (A) tail maintains a defined length and sequence, preventing shortening due to deletion mutations during large-scale fermentation.
3Ease of manufacture
If conventional poly (A) tail coding sequence is used, then in vitro transcription can proceed, but the sequence is prone to deletion mutations during large scale fermentation
Solution Approach 1:
The poly (A) tail coding sequence is segmented into distinct functional elements (element a, b, c, d) with specific nucleotide compositions and length ranges. This segmentation prevents deletion mutations during E. coli replication while maintaining the sequence's capability to direct poly (A) tail addition during in vitro transcription. The segmented structure allows the sequence to be reliably propagated in large-scale fermentation.
Solution Approach 2:
The poly (A) tail coding sequence is constructed as a composite of different nucleotide elements with specific properties. Element a provides the core poly (A) sequence, element b provides additional A nucleotides with controlled length, element c provides non-A nucleotide interruptions, and element d provides terminal sequences with specific constraints. This composite structure enhances replication stability in E. coli while maintaining in vitro transcription capability.
Data Source
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AI summary
An engineered DNA molecule capable of being replicated in a cell, comprising a poly (A) tail coding sequence that makes the engineered DNA molecule more conservative when replicated in cells, particularly in prokaryotic cells, while adjusting the expression level of RNA in eukaryotic cells. Also provided are an RNA comprising the poly (A) tail and a use thereof.