Circularizing Double-Stranded DNA via Enzymatic Nicking and Ligation
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Solution Overview
Problem
Current methods for generating circularized double-stranded nucleic acids often result in low yields and require bacterial systems for amplification, limiting efficiency and versatility.
Innovation Solution
A method involving a linear or circular double-stranded DNA nucleic acid with specific structural components, including homologous and non-homologous regions, is used to achieve high-yield circularization through a series of enzymatic steps, including digestion, nicking, and ligation, without the need for bacterial amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional methods (PCR, restriction enzymes, ligases) are used to assemble nucleic acids into circular vectors, then circular nucleic acids can be produced, but the yield is poor (10% at most) and requires bacterial systems for amplification
Solution Approach 1:
The method divides the circularization process into distinct enzymatic steps: (1) digestion with type II restriction enzyme to generate sticky ends, (2) nicking with type I restriction enzyme to create single-strand breaks, (3) annealing of complementary overhangs, and (4) ligation to seal the circle. This segmentation allows each step to be optimized independently, achieving high yield without requiring complex bacterial systems.
Solution Approach 2:
The patent uses type I restriction enzymes as intermediary tools that specifically create nicks in the DNA strand without completely digesting the molecule. These nicks serve as intermediaries that enable subsequent ligation to form circular structures. The type I enzyme acts as a mediator between the linear DNA and the final circular product, enabling circularization with high efficiency.
2Productivity
If bacterial systems are used to amplify circular nucleic acids, then higher yields can be achieved, but the process becomes more complex and time-consuming
Solution Approach 1:
The method enables self-assembly of circular nucleic acids through in vitro enzymatic reactions without requiring bacterial host cells. The DNA molecule itself serves as the substrate that undergoes circularization through controlled enzymatic actions, eliminating the need for bacterial amplification systems. This self-service approach achieves high yields directly in the test tube, saving time and simplifying the process.
3Productivity
If multiple-molecule DNA is used to form circular structures, then circular nucleic acids can be produced, but the yield remains limited and requires complex assembly
Solution Approach 1:
The method performs preliminary actions by first generating sticky ends through type II restriction enzyme digestion, then creating specific nicks through type I enzyme action before final ligation. This preliminary preparation of the DNA structure enables straightforward circularization in a single in vitro reaction, avoiding the need for complex multi-step assembly procedures required when using multiple-molecule 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
This method enables efficient circularization of double-stranded DNA with yields up to 90% efficiency, surpassing traditional methods, and allows for functionalization and various applications such as gene therapy and protein production.
Implementation Method 1
digesting the circular, linear or linearized nucleic acid in the presence of (i) a polypeptide with nickase activity capable of introducing a nick within TBC
Implementation Method 2
recessing both ends of identical orientation of the nucleic acid obtained in step b1) or step b2), in the presence of a polypeptide having a 3'-5' nuclease activity
Implementation Method 3
annealing the DNA nucleic acid obtained at step c), thereby generating 2 gaps, 2 nicks, or 1 nick and 1 gap
Implementation Method 4
filling the 1 or 2 gaps generated at step d), step e) being optional when 2 nicks are generated at step d
Implementation Method 5
sealing at least one nick, so as to obtain a circularized double stranded DNA nucleic acid
Data Source
AI summary
A method, in particular an in vitro method, for the circularization of a double stranded DNA nucleic acid. Also, a circularized double stranded DNA nucleic acid obtainable by the method. Another aspect pertains to a host cell comprising a circularized double stranded DNA nucleic acid obtainable by the method. Further, therapeutic and non-therapeutic uses of a circularized double stranded DNA nucleic acid obtainable by the method. Finally, a kit for the circularization of a double stranded DNA nucleic acid.