Linear Double-Stranded DNA Tagging for Reusable RNA Transcription
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Solution Overview
Problem
Current methods for producing RNA in vitro transcription are inefficient and costly, particularly at an industrial scale, and lack a directed, non-PCR-based method for coupling DNA templates to a support or tag, which can impair RNA polymerase efficiency and require DNA destruction post-use.
Innovation Solution
The development of linear double stranded DNA with a coding sequence element coupled to a support or tag at the 3′ end of its non-coding strand, using methods such as click chemistry or enzymatic tagging, allowing for efficient and reusable DNA templates in RNA in vitro transcription.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If DNA templates are coupled to support or tag using current methods, then DNA can be separated from RNA product, but DNA must be destroyed post-use by DNAse digestion which increases process complexity and cost
Solution Approach 1:
The patent implements a system where DNA templates are coupled to magnetic beads via a tag, allowing the DNA to be recovered and reused after RNA transcription. The magnetic beads enable easy separation and recovery of the DNA template from the reaction mixture, eliminating the need for DNA destruction and enabling multiple uses of the same DNA template.
Solution Approach 2:
The patent introduces a tag as an intermediary element that couples the DNA template to the magnetic bead support. This tag-mediated coupling system allows for controlled attachment and detachment of DNA to the support, facilitating reuse while maintaining the ability to separate DNA from the RNA product through magnetic separation.
2Ease of manufacture
If PCR-based methods are used to couple DNA to support, then coupling can be achieved, but the process becomes error-prone and sequence-dependent
Solution Approach 1:
The patent extracts the coupling process from PCR-based methods and implements a direct enzymatic tagging approach. This eliminates the errors and sequence dependencies inherent in PCR by using a straightforward enzymatic reaction to attach the tag to the DNA template, which then couples to the magnetic bead support without requiring amplification steps.
Solution Approach 2:
The patent replaces the complex PCR-based mechanical coupling system with a simpler enzymatic tagging system. Instead of using PCR amplification and mechanical coupling steps, the invention uses enzymatic reactions to attach tags directly to DNA, which then bind to magnetic beads through specific interactions, reducing errors and improving reliability.
3Productivity
If DNA templates are used for RNA in vitro transcription, then RNA production can be achieved, but DNA must be removed from final product by digestion and purification which increases time and cost
Solution Approach 1:
The patent uses magnetic beads as an intermediary to maintain physical separation between the DNA template and the final RNA product throughout the transcription process. The DNA remains attached to the magnetic beads while the RNA is released into solution, eliminating the need for post-transcription DNA removal steps and allowing immediate purification of the RNA product.
Solution Approach 2:
The patent segments the transcription system into two distinct phases: the DNA template remains bound to the magnetic bead support while the RNA product is released into solution. This spatial segmentation allows the DNA to stay in place during transcription and facilitates easy separation of the RNA product without requiring DNA digestion or additional purification steps.
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 high-quality RNA production with defined lengths and efficient separation from DNA templates, suitable for industrial-scale RNA production without DNA destruction, ensuring safety and reducing costs.
Implementation Method 1
adding a modified deoxynucleotide to the 3′ ends of linear double stranded DNA and coupling said modified deoxynucleotide to a support or tag
Implementation Method 2
Digestion of the obtained linear double stranded DNA by an endonuclease leads to linear double stranded DNA which comprises a support or a tag only at the 3′ end of its non-coding strand
Data Source
AI summary
The present invention is concerned with linear double stranded DNA, which is coupled to a single support or a tag at the 3′ end of its non-coding strand and methods for producing said linear double stranded DNA. The present invention further relates to the use of said linear double stranded DNA in an RNA in vitro transcription reaction and also to a method for producing RNA in vitro. The present invention also relates to a bioreactor for RNA in vitro transcription.