In Vitro Circular DNA Synthesis Without Bacterial Sequences
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Solution Overview
Problem
Existing methods for synthesizing circular DNA molecules, particularly supercoiled circular dsDNA, are inefficient, costly, and require multiple steps, often incorporating bacterial DNA sequences that can trigger immune responses and are difficult to purify, limiting their clinical use.
Innovation Solution
A method involving the ligation of stem-loop or hairpin DNA molecules using DNA ligase and exonuclease to create circular single-stranded DNA, followed by conversion to supercoiled double-stranded DNA using recombinase and gyrase, eliminating bacterial DNA sequences and simplifying purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
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 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 a minimized plasmid that eliminates immunogenic elements while preserving the desired therapeutic function, directly resolving the contradiction between ease of production and immune response risk
Solution Approach 2:
The patent employs a disposable E. coli production system where plasmids are rapidly produced and then extracted. The bacterial production machinery is used temporarily for manufacturing, then discarded, while the final product is a purified, bacteria-free DNA molecule. This allows easy manufacturing without carrying over bacterial contaminants that cause immune responses
2Reliability
If antibiotic resistance-encoding genes are included in plasmids for selection, then they enable propagation and selection in E. coli host strains, but they can be transferred to bacteria in the human microbiome and trigger immune reactions
Solution Approach 1:
The patent removes antibiotic resistance-encoding genes from the final plasmid structure after they have served their purpose during E. coli propagation and selection. The selection capability is utilized during manufacturing, then the harmful selection markers are extracted and discarded, leaving a clean therapeutic plasmid without gene transfer risk
Solution Approach 2:
The patent performs selection and propagation operations preliminarily during the manufacturing phase using antibiotic resistance genes. Once the desired plasmid population is obtained, the antibiotic resistance genes are removed before clinical application. This preliminary use of selection markers enables reliable manufacturing without carrying forward the harmful gene transfer risk to patients
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 employs circularization of linear DNA molecules to form closed circular plasmids. This curvature transformation creates supercoiled structures that are more compact and resistant to degradation. The circular form enables supercoiling which enhances transfection efficiency and nuclear localization while maintaining the simplicity of initial linear DNA synthesis
Solution Approach 2:
The patent changes the topological state of DNA from linear to circular, and from relaxed to supercoiled form. This parameter change in DNA conformation dramatically improves transfection efficiency and stability while preserving the ease of initial synthesis. The supercoiled state provides additional protection from shear forces and promotes nuclear entry
4Shape
If in vitro ligation of linear DNA molecules is used to produce circular ssDNA, then it can create circular structures, but the yield of producing large size ss circular DNA is low
Solution Approach 1:
The patent performs preliminary amplification of linear DNA templates using PCR or rolling circle amplification before the ligation step. This generates abundant linear DNA substrates that dramatically improve the yield of subsequent circularization reactions. The preliminary amplification ensures sufficient material is available for efficient ligation to form large-size circular ssDNA molecules
Solution Approach 2:
The patent uses splint oligonucleotides as intermediary molecules that facilitate the ligation reaction. These splints hybridize to the linear DNA ends and bring them into proximity for efficient ligation by DNA ligase. The intermediary splint structure enables high-yield circularization even for large-size DNA molecules that would otherwise be difficult to ligate efficiently
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
This method produces large quantities of high-purity, supercoiled circular DNA molecules without bacterial sequences, reducing production costs and immune response risks, and enabling scalable synthesis for therapeutic applications.
Implementation Method 1
The subject invention provides methods, compositions and kits for efficiently synthesizing closed circular single-stranded nucleic acids and double-stranded nucleic acids of various sizes and sequences. The principle of the method for efficiently synthesizing closed circular single-stranded nucleic acids and double-stranded nucleic acids is to ligate two stem-loop or hairpin DNA molecules into a circular single-stranded DNA molecule.
Implementation Method 2
A weakness of this method is the low yield of producing large size ss circular DNA. Another method is the use of phage M13 or phagemids to produce ss circular DNA molecules. A drawback of this method is that certain bacterial DNA sequences, such as phage M13 replication origin, are still required and may cause issues for the following applications.
Implementation Method 3
In rolling circle DNA amplification, the circular ssDNAs, in combination with short strands of single-stranded complementary primer DNAs, are employed as templates for replication by DNA polymerase, which provides concatemers containing tens to hundreds of tandem repeats and has been widely adopted for various purposes.
Implementation Method 4
Because (−) supercoiled DNA is the physiologically preferred form of DNA for the transfection of mammalian cells, linear dsDNA molecules, which cannot be supercoiled, may limit their potential for clinical use. Negative supercoiling makes DNA more compact, which promotes nuclear localization and provides additional protection from the shear forces of aerosolization.
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.