Seamless Nucleic Acid Assembly via Enzymatic Cascade
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for nucleic acid synthesis are limited by scalability, automation, speed, accuracy, and cost, making efficient seamless nucleic acid assembly challenging.
Innovation Solution
The methods involve providing a plurality of polynucleotides and mixing them with an exonuclease, a flap endonuclease, a polymerase, and a ligase, where the polynucleotides are annealed in a processive predetermined order based on complementary sequences, using specific concentrations of enzymes like exonuclease III and flap endonuclease 1.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional nucleic acid synthesis methods are used, then synthesis can be performed on a small scale, but scalability, automation, speed, and cost-effectiveness deteriorate
Solution Approach 1:
The patent combines multiple enzymatic functions (exonuclease, flap endonuclease, polymerase, and ligase activities) into a single reaction mixture, allowing simultaneous processing of polynucleotide fragments. This merging of functions enables seamless assembly of multiple fragments in one reaction, dramatically improving productivity while maintaining manageable complexity through a unified protocol
Solution Approach 2:
The method employs a universal enzyme cocktail that can process various types of polynucleotide fragments with different sequences and lengths. The reaction system is designed to accommodate multiple fragments simultaneously, making the method universally applicable to different assembly scenarios without requiring method redesign, thus improving scalability and productivity
2Extent of automation
If manual nucleic acid assembly methods are used, then process control is possible, but automation and speed deteriorate
Solution Approach 1:
The enzymatic reaction system is designed to self-assemble polynucleotide fragments in the correct order through complementary base pairing and enzymatic processing. The exonuclease creates single-stranded overhangs, the flap endonuclease processes flaps, the polymerase fills gaps, and the ligase seals nicks - all occurring automatically in the reaction mixture without manual intervention, enabling full automation while maintaining high precision through built-in enzymatic fidelity
Solution Approach 2:
The method incorporates inherent feedback mechanisms where the enzymatic reactions are dependent on the presence and state of the polynucleotide fragments. The exonuclease only acts on specific ends, the flap endonuclease only processes formed flaps, and the ligase only seals completed nicks, creating a self-regulating system that ensures accurate assembly while allowing automated execution
3Speed
If rapid nucleic acid synthesis is pursued, then speed improves, but accuracy and completeness of assembly deteriorate
Solution Approach 1:
The patent employs a continuous enzymatic cascade where exonuclease processing, flap endonuclease action, polymerase filling, and ligase sealing occur in continuous sequence without interruption. This continuous action allows rapid progression through all assembly steps while maintaining accuracy, as each enzymatic step completes before the next begins, ensuring completeness and correctness at high speed
Solution Approach 2:
The method performs preliminary processing of polynucleotide fragments before assembly, including exonuclease treatment to create appropriate ends and flap endonuclease processing to prepare flaps. These preliminary actions ensure that fragments are pre-conditioned for accurate and rapid assembly, enabling high speed without sacrificing precision by resolving potential issues before the main assembly reaction
4Productivity
If efficient enzyme concentrations are used, then assembly efficiency improves, but cost increases
Solution Approach 1:
The patent optimizes enzyme concentrations to achieve maximum assembly efficiency at minimal effective doses. By carefully adjusting the parameters of enzyme amounts, reaction time, and temperature, the method achieves high productivity while minimizing the quantity of expensive enzymes required, thus improving the cost-effectiveness ratio
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 approach enables efficient and accurate nucleic acid synthesis and assembly, improving scalability, automation, speed, and reducing costs, while maintaining high accuracy and efficiency.
Implementation Method 1
mixing the plurality of polynucleotides with an exonuclease, a flap endonuclease, a polymerase, and a ligase
Implementation Method 2
mixing the plurality of polynucleotides with an exonuclease, a flap endonuclease, a polymerase, and a ligase
Implementation Method 3
mixing the plurality of polynucleotides with an exonuclease, a flap endonuclease, a polymerase, and a ligase
Implementation Method 4
mixing the plurality of polynucleotides with an exonuclease, a flap endonuclease, a polymerase, and a ligase
Implementation Method 5
the plurality of polynucleotides are annealed in a processive predetermined order based on a complementary sequence between adjacent polynucleotides
Data Source
AI summary
Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. Methods, systems, and compositions as provided herein provide for efficient assembly of nucleic acids without primer removal. Methods, systems, and compositions for seamless nucleic acid assembly comprise use of an endonuclease or exonuclease, optionally in conjunction with additional enzymes to assemble nucleic acids or polynucleotides.