3′-Stabilizing mRNA Tail Structure for DNA-Free Protein Expression
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Solution Overview
Problem
Existing methods of protein expression in cells face challenges such as integration of heterologous DNA into host genomic DNA, leading to alterations and damage, and require multiple processing steps that create lag times before protein generation, particularly in primary cells or modified cell lines, with difficulties in obtaining DNA expression at reasonable rates or concentrations.
Innovation Solution
The development of polynucleotides with a specific structure incorporating a 5'-cap, 5'-UTR, coding region, and a 3'-stabilizing region containing alternative nucleosides like inverted thymidine, linked by a specific linker, to enhance intracellular translation and stabilization of mRNA molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If heterologous DNA is introduced into cells for protein expression, then protein production is achieved, but DNA integrates into host genomic DNA causing alterations and damage
Solution Approach 1:
The invention segments the genetic material into separate functional components: a DNA template for in vitro transcription and a stabilized mRNA product for translation. This separation prevents the DNA from integrating into host genomic DNA while still enabling protein expression through the isolated mRNA molecule.
Solution Approach 2:
The invention introduces an intermediary step of in vitro transcription, where DNA is converted to mRNA in a controlled external environment before entering the cell. This intermediary process prevents direct DNA-cell interaction and genomic integration, while the mRNA serves as the mediating carrier of genetic information.
2Productivity
If multiple processing steps are used for protein expression (DNA transport to nucleus, transcription to RNA, RNA transport to cytoplasm, translation to protein), then complete protein synthesis is achieved, but lag times occur before protein generation
Solution Approach 1:
The invention performs preliminary transcription of DNA to mRNA in vitro before introducing the mRNA into the cell. This preliminary action eliminates the need for intracellular transcription and nuclear transport steps, allowing translation to begin immediately upon mRNA entry and significantly reducing lag time.
Solution Approach 2:
The invention extracts the transcription process from the intracellular environment and performs it externally in vitro. This removal of the transcription step from the cellular pathway eliminates the associated time delays while maintaining the essential function of converting genetic information to a translatable form.
3Productivity
If DNA is introduced into primary cells or modified cell lines for expression, then protein production is attempted, but expression is difficult to obtain at reasonable rates or concentrations
Solution Approach 1:
The invention uses stabilized mRNA as an intermediary that bypasses the problematic DNA integration and expression steps. The mRNA can be directly translated by ribosomes without requiring nuclear import, chromatin remodeling, or transcriptional activation, making expression achievable in difficult-to-transfect cell types at higher rates.
Solution Approach 2:
The invention changes the chemical parameters of the nucleic acid by incorporating modified nucleosides and stabilizing elements into the mRNA structure. These parameter changes increase the mRNA's stability and translation efficiency, enabling reasonable expression rates in primary cells and modified cell lines that would otherwise poorly express exogenous DNA.
Data Source
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AI summary
The invention features polynucleotides encoding a polypeptide including a 3'-stabilizing region and having increased stability compared to wild-type polynucleotides.