Scarless Nucleic Acid Assembly via Stem-Loop Ligation
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Solution Overview
Problem
Current recombinant nucleic acid and genetic engineering methodologies are inefficient due to issues such as large scars between Biological Parts (BPs), limited assembly capabilities, and the need to avoid restriction enzyme recognition sites, which hinder the construction of plasmids in molecular biology.
Innovation Solution
A method involving the ligation of nucleic acid molecules with defined overhangs that form stem-loop structures, allowing for scarless assembly of multiple fragments without the need for restriction enzyme sites, using phosphorothioate oligos and chemical cleavage to create 1-nt sticky ends, and employing stem-loop oligos to barcode fragments for efficient assembly.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional restriction enzyme-based assembly methods are used, then assembly of nucleic acid fragments can be achieved, but large scars (conserved useless nucleotides) are left between Biological Parts affecting biological function
Solution Approach 1:
The invention extracts and removes the harmful restriction enzyme recognition sites and scar sequences from the assembly process. By using homology-directed assembly with overlapping ends, the method eliminates the need for restriction sites, thereby removing the source of scar formation and preserving the functional integrity of Biological Parts.
Solution Approach 2:
The invention changes the assembly mechanism from restriction enzyme-based (recognizing specific sequences) to homology-based (recognizing complementary overlapping sequences). This parameter change in the recognition mechanism allows scarless assembly by using the homology regions themselves for joining without leaving conserved useless nucleotides.
2Ease of manufacture
If BioBricks assembly method is used, then standardized Biological Parts can be assembled, but only two BPs can be assembled in one round resulting in long plasmid construction time
Solution Approach 1:
The invention merges multiple assembly capabilities into a single reaction system. By using homology-directed assembly with appropriately designed overlapping ends, multiple Biological Parts can be assembled in one round, combining the benefits of standardization with increased assembly capacity and reduced construction time.
Solution Approach 2:
The invention incorporates preliminary design of homology regions and overhangs in the Biological Parts. These pre-designed features enable direct assembly without intermediate steps, allowing multiple fragments to be joined in a single reaction round while maintaining standardization.
3Adaptability or versatility
If Golden gate or BASIC assembly methods are used, then multi-fragment assembly is enabled, but certain restriction enzyme recognition sites must be avoided in sequence of BPs
Solution Approach 1:
The invention substitutes the mechanical restriction enzyme recognition system with a homology-based molecular recognition system. This replacement eliminates the constraint of avoiding restriction sites, as the assembly is directed by complementary homology sequences rather than enzyme recognition sites, thereby removing the harmful constraint on Biological Parts design.
4Manufacturing precision
If customized Biological Parts are synthesized from scratch, then specific genetic sequences can be obtained, but commercial synthesis and delivery takes time and increases cost
Solution Approach 1:
The invention performs preliminary design of homology regions and assembly interfaces in the Biological Parts. This pre-prepared structure allows rapid assembly once the parts are available, reducing the time loss associated with commercial synthesis and delivery by enabling immediate assembly without additional processing steps.
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
Enables efficient, scarless assembly of multiple nucleic acid fragments into functional plasmids, reducing construction time and cost, and allowing for flexible recombination of genetic parts, thereby improving the efficiency of plasmid construction in molecular biology.
Implementation Method 1
providing a second nucleic acid molecule capable of forming a stem-loop structure with an overhang of at least one nucleotide; wherein the overhang of the second nucleic acid molecule is substantially complementary to the first overhang of the first end of the first nucleic acid molecule
Implementation Method 2
ligating the first nucleic acid molecule to the second nucleic acid molecule at the complementary overhangs to form a single nucleic acid molecule
Data Source
AI summary
The present invention relates to ligation and/or assembly of nucleic acid molecules. Particularly, a double-stranded target nucleic acid having overhangs of at least one nucleotide is ligated with another nucleic acid molecule capable of forming a stem-loop structure with an overhang of at least one nucleotide. The invention is suitable for tagging nucleic acid molecules. In specific embodiments, the overhangs can be produced by chemical cleavage of phosphorothioate-modified nucleic acid molecules. The invention further relates to the amplification of the ligated product, and using the resultant amplicon for assembly of multiple nucleic acid fragments.