Directed Assembly of Synthetic Nucleic Acids via Masking
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current gene synthesis methods face challenges in efficiently assembling synthetic nucleic acid sequences in vitro, particularly due to conflicting nucleotide sequences that interfere with protocols, such as restriction enzyme sites or sequences that hinder hybridization during polymerase chain assembly.
Innovation Solution
A method involving the production of a complex by annealing nucleic acids with unique sequences that do not hybridize, followed by multiple rounds of primer extension and circularization, and the use of Type IIS restriction endonucleases to mask and remove conflicting sequences, allowing for the synthesis of synthetic nucleic acids by polymerase chain assembly and subsequent re-ligation to restore the target sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If classical cloning and mutagenesis procedures are used for gene synthesis, then the process follows traditional methods, but it is more expensive and less economical
Solution Approach 1:
The target nucleic acid sequence is divided into multiple overlapping oligonucleotide fragments that can be independently synthesized and then assembled together through hybridization and extension reactions, enabling economical de novo synthesis without classical cloning
Solution Approach 2:
The oligonucleotide fragments self-assemble through complementary hybridization and primer extension reactions, where each fragment serves as both a product and a template for other fragments, eliminating the need for traditional cloning procedures
2Productivity
If conflicting nucleotide sequences are present in the target sequence, then the target sequence can be synthesized, but the conflicting sequences interfere with protocols such as restriction enzyme digestion or hybridization
Solution Approach 1:
Masking sequences are inserted into conflicting nucleotide sequences to temporarily remove or hide the interfering elements during assembly, allowing the target sequence to be synthesized without interference, and the masking sequences are later removed by restriction enzyme digestion
Solution Approach 2:
Masking sequences act as intermediary elements that are introduced into conflicting regions to prevent interference during hybridization and assembly processes, and are subsequently removed to restore the original target sequence
3Manufacturing precision
If unique sequences are used for annealing and assembly, then nucleic acids can be directed to assemble correctly, but the unique sequences must not hybridize with each other which limits sequence design
Solution Approach 1:
Unique sequences are designed with specific local properties (complementarity patterns, GC content, length) that enable directed hybridization and assembly accuracy while maintaining overall sequence design flexibility for different target sequences
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 assembly and synthesis of synthetic nucleic acids by overcoming interference from conflicting sequences, facilitating the production of synthetic genes and allowing for the creation of circularized products with specific sequences, enhancing the precision and efficiency of gene synthesis.
Implementation Method 1
annealing: (i) a first nucleic acid comprising, in order, a first unique sequence, a first central sequence and a second unique sequence; and (ii) a second nucleic acid comprising, in order, the second unique sequence, a second central sequence and a third unique sequence
Implementation Method 2
subjecting the first complex to multiple rounds of primer extension to extend the first and second nucleic acids using each other as a template
Implementation Method 3
the masking sequence comprises recognition sites for one or more Type IIS restriction endonucleases and digestion of the disrupted target sequence by the one or more Type IIS restriction endonucleases
Data Source
AI summary
Provided herein is a method comprising: producing a first complex by annealing: a first nucleic acid comprising, in order, a first unique sequence, a first central sequence and a second unique sequence; and a second nucleic acid comprising, in order, said second unique sequence, a second central sequence and a third unique sequence; wherein the first, second and third unique sequences do not hybridize with each other; subjecting said first complex to multiple rounds of primer extension to extend the first and second nucleic acids using each other as a template, thereby producing a first product molecule that contains, in order, the first unique sequence, the first central sequence, the second unique sequence, the second central sequence and the third unique sequence; and circularizing said first product molecule by intramolecularly ligating the ends of said product molecule together. Kits and compositions relating to the method are also provided.