Universal Plasmid Backbone With Multi-Enzyme Landing Sites for mRNA Production
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Solution Overview
Problem
Existing methods for producing mRNA-based drugs are inefficient and costly due to the need for custom plasmids/vectors for each new gene of interest (GOI), and there is a lack of flexible tools for nucleic acid amplification and modification that preserve essential functions.
Innovation Solution
A universal landing platform (ULP) DNA molecule with multiple type II restriction enzyme sites, allowing for targeted editing and amplification without disrupting critical sequences, compatible with in vitro transcription and eukaryotic cell expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a new restriction enzyme is selected for each new GOI to ensure the enzyme does not cut on the GOI, then the specificity of restriction enzyme digestion is improved, but the time and cost of production increases drastically
Solution Approach 1:
The patent applies universality by designing a single plasmid backbone containing multiple different restriction enzyme sites that can all be used for cloning any GOI. Instead of selecting a new enzyme for each GOI, the same plasmid with its multiple restriction sites serves universal purposes for different genes, reducing the need to build new plasmids repeatedly.
Solution Approach 2:
The plasmid is pre-designed with multiple restriction enzyme sites incorporated into its backbone before use. This preliminary preparation ensures that when a GOI is ready for cloning, multiple compatible restriction sites are already available, eliminating the time-consuming process of selecting and validating new enzymes for each gene.
2Reliability
If a new plasmid is built for each new GOI to ensure the enzyme does not cut on the GOI, then the compatibility of restriction enzyme digestion is improved, but the cost and complexity of production increases
Solution Approach 1:
A single universal plasmid backbone is designed with multiple restriction enzyme sites that can accommodate different GOIs. This universal plasmid reduces the complexity of building new plasmids for each gene, as the same backbone can be reused with different inserts.
Solution Approach 2:
The plasmid is segmented into a stable backbone containing multiple restriction sites and a variable insert region for the GOI. This segmentation allows the backbone to be reused while only the insert needs to be changed, simplifying the overall construction process.
3Adaptability or versatility
If multiple restriction enzyme sites are included in a universal plasmid backbone, then the versatility and efficiency of nucleic acid amplification is improved, but the risk of unintended cutting of the GOI increases
Solution Approach 1:
The plasmid backbone is designed with multiple restriction sites located in specific regions that are distinct from where the GOI will be inserted. This local placement ensures that while multiple enzymes are available for cloning, the GOI sequence itself does not contain these restriction sites and thus cannot be inadvertently cut.
Solution Approach 2:
The plasmid backbone acts as an intermediary that provides multiple restriction sites for cloning purposes, while the GOI is inserted into a separate region. This intermediary structure allows the restriction sites to serve their function without directly interacting with or cutting the GOI sequence.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables efficient and cost-effective production of mRNA by allowing multiple restriction enzymes to target the same site, reducing production time and costs while maintaining functional integrity.
Implementation Method 1
a type II restriction enzyme is often used. The problem is that this class of restriction enzyme must not cut the GOI.
Data Source
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AI summary
The present invention relates to a DNA molecule comprising a nucleic acid sequence comprising at least 3 restriction enzymes sites, preferably at least 4 restriction enzymes sites, as well as vectors or cells comprising thereof.