Engineered DNA Poly(A) Tail Coding for Stable E. coli Replication
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Solution Overview
Problem
The instability of poly (A) tails during replication in prokaryotic systems, such as E. coli, leads to deletion mutations, affecting the in vivo stability and biological activity of mRNA drugs, which is a challenge in the preparation process of in vitro transcription template plasmids for large-scale fermentation.
Innovation Solution
An engineered DNA molecule with a specific poly (A) tail coding sequence, comprising elements a, b, c, and d, is designed to enhance replication stability and regulatory control over RNA expression levels, ensuring the poly (A) tail is conserved during in vitro preparation and in eukaryotic cell expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If a poly (dA:dT) repeat sequence is included in the template plasmid to achieve co-transcriptional addition of poly (A) tail, then the poly (A) tail can be added downstream of the 3′ UTR in a co-transcriptional manner, but the poly (dA:dT) repeat sequence is unstable during replication in E. coli with deletion mutations often occurring, leading to shortening of poly(dA:dT)
Solution Approach 1:
The poly (A) tail coding sequence is divided into multiple segments: a long stretch of adenine nucleotides (element a, ≥20 nt), short adenine elements (element b, 3-10 nt), and interrupting non-A nucleotide elements (element c, T/C/G). This segmentation prevents the formation of unstable long homopolymeric repeats while still enabling co-transcriptional poly (A) tail addition, thereby resolving the contradiction between ease of manufacture and compositional stability.
Solution Approach 2:
The invention introduces non-A nucleotide elements (T, C, or G) at specific positions within the poly (A) tail coding sequence to locally disrupt the homopolymeric adenine repeat. This local modification maintains the overall poly (A) function while preventing deletion mutations during E. coli replication, thus achieving both co-transcriptional addition capability and replication stability.
2Ease of manufacture
If poly (A) tail is synthesized by post-transcriptional modification under the action of typical poly (A) polymerase in eukaryotic cell, then the poly (A) tail can be added after transcription, but this process is not conducive to the preparation process of in vitro transcription template plasmids through large scale fermentation
Solution Approach 1:
The poly (A) tail coding sequence is pre-installed in the DNA template plasmid as a poly (dA:dT) repeat sequence before in vitro transcription. During transcription, RNA polymerase automatically synthesizes the poly (A) tail co-transcriptionally without requiring subsequent poly (A) polymerase treatment. This preliminary incorporation of the poly (A) coding sequence enables large-scale plasmid fermentation and automated mRNA production, resolving the contradiction between manufacturing ease and processing time.
Data Source
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.