Cell-Free Circular Nucleic Acid Amplification With Rolling Circle
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Solution Overview
Problem
Current methods for producing synthetic nucleic acid molecules are costly, require expensive equipment, and result in impure products due to cell-mediated amplification, with difficulties in controlling fidelity and expression.
Innovation Solution
A cell-free method for amplifying synthetic circular nucleic acids using rolling circle amplification, endonucleases, ligases, and topoisomerases to produce highly pure products, eliminating the need for cell-based systems and reducing impurities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If circular nucleic acid is produced using traditional cloning methods in living cells, then the nucleic acid can be obtained, but the process requires complex living cell systems and extensive manual intervention
Solution Approach 1:
The patent replaces the biological system (living cells) with a chemical system (cell-free transcription-translation system). The in vitro system uses purified enzymes, nucleotides, and synthetic templates to produce circular nucleic acids without requiring living organisms, thereby simplifying the manufacturing process while maintaining production capability.
Solution Approach 2:
The patent divides the production process into distinct modular components: synthetic double-stranded DNA templates, in vitro transcription mix (containing RNA polymerase, nucleotides, and buffers), and ligation reagents. This segmentation allows each component to be optimized independently and simplifies the overall manufacturing workflow.
2Productivity
If traditional cloning methods are used, then circular nucleic acid can be produced, but the process is time-consuming and requires multiple manual steps
Solution Approach 1:
The patent implements a continuous in vitro transcription-translation process where synthetic DNA templates are transcribed into RNA and translated into circular nucleic acids in a single uninterrupted reaction system. This eliminates the need for multiple discrete manual steps and intermediate handling, significantly reducing production time and increasing efficiency.
Solution Approach 2:
The patent uses pre-synthesized double-stranded DNA templates that are prepared in advance with specific sequences designed to produce the desired circular nucleic acid products. This preliminary preparation of templates with optimized sequences accelerates the subsequent transcription and translation steps.
3Stability of the object's composition
If linear nucleic acid intermediates are used in traditional methods, then circular nucleic acid can be formed, but linear intermediates are more susceptible to degradation and have lower stability
Solution Approach 1:
Instead of producing linear nucleic acid intermediates and then circularizing them (the traditional approach), the patent inverts the process by directly producing circular nucleic acids through in vitro transcription and translation. This inversion eliminates the unstable linear intermediate step entirely, as the circular structure is formed during the translation process itself.
Solution Approach 2:
The patent converts the potential harm of linear intermediate degradation into a benefit by designing a system where the circular structure is formed directly during translation. The circular configuration provides inherent stability and resistance to degradation, turning what would have been a vulnerable intermediate into a stable final product.
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
The method provides cost-effective, high-fidelity amplification of synthetic nucleic acids with streamlined purification, producing pure and efficient quantities suitable for therapeutic and research applications.
Implementation Method 1
an in vitro transcription-translation system is used
Implementation Method 2
an in vitro transcription-translation system is used
Data Source
Figure 1
Figure 2A~2B
Figure 3A~3D
AI summary
The present disclosure relates to a process of cell-free circular nucleic acid amplification using rolling circle amplification.