In Vitro Enzymatic Circular DNA Synthesis Without Bacterial Sequences
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Solution Overview
Problem
Current methods for synthesizing circular DNA molecules, such as plasmid DNA and minicircles, are limited by high contamination, cost, and the need for bacterial DNA sequences, which can trigger immune responses and are difficult to remove, and existing methods are complex and time-consuming.
Innovation Solution
A method involving the ligation of stem-loop or hairpin DNA molecules to form circular single-stranded and double-stranded nucleic acids using enzymatic systems, including DNA ligase and exonuclease, to produce supercoiled circular DNA molecules without bacterial DNA sequences, allowing for scalable and efficient synthesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If plasmid DNA molecules are constructed and produced in E. coli cells, then they are heat stable and easy to store and transport, but they contain bacterial DNA sequences that increase plasmid size and may lead to immune response and gene silencing
Solution Approach 1:
The patent extracts and removes bacterial DNA sequences (origin of replication, antibiotic resistance genes) from the plasmid structure, retaining only the essential therapeutic gene cassette. This creates minicircles that eliminate immune response and gene silencing while preserving the therapeutic function and storage stability of plasmid DNA.
Solution Approach 2:
The patent segments the plasmid DNA into two parts: essential therapeutic elements (gene cassette) and non-essential bacterial elements (origin of replication, antibiotic resistance genes). By separating and removing the bacterial elements, the invention creates a purified therapeutic DNA molecule that maintains stability but eliminates immunogenicity.
2Object-affected harmful factors
If DNA minicircles are generated in vivo using site-directed recombination, then they consist almost entirely of the target gene cassette without bacterial DNA sequences, but parent plasmid contamination is still high and production cost is very high
Solution Approach 1:
The patent uses PCR to amplify the target gene cassette from the plasmid template, creating multiple copies of the pure therapeutic sequence without requiring complete minicircle assembly in vivo. This in vitro copying approach eliminates parent plasmid contamination and reduces production cost by using simple, scalable PCR technology.
Solution Approach 2:
The patent replaces the complex in vivo site-directed recombination system with a simpler in vitro PCR-based approach. This substitution eliminates the need for bacterial transformation, recombination enzyme expression, and multiple purification steps, thereby reducing production cost and improving yield purity.
3Ease of manufacture
If linear dsDNA molecules are used for transfection, then they are simpler to synthesize, but they cannot be supercoiled and may limit their potential for clinical use
Solution Approach 1:
The patent converts linear dsDNA molecules into circular minicircle structures. This curvature transformation enables the DNA to adopt supercoiled conformations that enhance transfection efficiency, nuclear localization, and protection from shear forces, while maintaining the synthesis simplicity of in vitro methods.
Solution Approach 2:
The patent changes the topological parameter of the DNA from linear to circular configuration. This parameter change enables supercoiling, which improves transfection reliability and clinical potential while preserving the ease of in vitro synthesis. The circular structure provides mechanical stability and resistance to degradation.
4Object-affected harmful factors
If in vitro ligation of linear DNA molecules is used to produce circular ssDNA, then bacterial DNA sequences can be avoided, but the yield of producing large size ss circular DNA is low
Solution Approach 1:
The patent performs preliminary PCR amplification to generate sufficient quantities of the target gene cassette before circularization. This preliminary action ensures that there is enough template material to overcome the low efficiency of in vitro ligation, thereby achieving high final yield of large-size circular ssDNA without bacterial sequences.
Solution Approach 2:
The patent uses PCR-amplified linear DNA as an intermediary product between the plasmid template and the final circular ssDNA. This intermediary step allows for amplification of the target sequence to high concentration, which then serves as abundant substrate for the low-efficiency ligation reaction, ultimately producing sufficient circular DNA.
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 produces high yields of pure circular DNA molecules without bacterial sequences, reducing immune response risks and production costs, and enables efficient applications like CRISPR/Cas9-mediated genome editing and therapeutic use.
Implementation Method 1
A method involving the ligation of stem-loop or hairpin DNA molecules to form circular single-stranded and double-stranded nucleic acids using enzymatic systems, including DNA ligase
Implementation Method 2
A method involving the ligation of stem-loop or hairpin DNA molecules to form circular single-stranded and double-stranded nucleic acids using enzymatic systems, including DNA ligase and exonuclease
Data Source
AI summary
A method, which synthesizes closed circular single-stranded and double-stranded DNA molecules using in vitro enzymatic systems, is described. Circular single-stranded DNA molecules and double-stranded DNA molecules (e.g., relaxed, or supercoiled) with various sizes can be synthesized. Unwanted DNA molecules, e.g., unligated oligomers, can be removed by exonucleases, such as T5 exonuclease, T7 exonuclease, lambda exonuclease, E. coli exonuclease I and/or III. A method of converting the single-stranded circular DNA molecules into double-stranded circular DNA molecules is also described. The single-stranded and double-stranded circular DNA molecules can be used in a variety of applications.