Nucleic Acid Splint Ligation for High-Yield Polynucleotide Synthesis
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Solution Overview
Problem
Existing ligation techniques for synthesizing long oligonucleotides or polynucleotides face inefficiencies and challenges in achieving high yield and accuracy, particularly in aligning the 3' end side of one nucleic acid with the 5' end side of another.
Innovation Solution
The use of nucleic acid splints that are complementary to the nucleic acid sequences and substantially free of deletions, allowing for improved ligation efficiency by aligning multiple polynucleotide fragment molecules to form a specific sequence, with enzymatic ligation reactions performed simultaneously or sequentially.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional ligation techniques are used to synthesize long oligonucleotides, then the process can proceed with standard methods, but the ligation efficiency is low and deletion errors occur frequently
Solution Approach 1:
The patent introduces nucleic acid splints as intermediary molecules that bridge the 3' end of one oligonucleotide and the 5' end of another. These splints are complementary to both fragments, forming a stable scaffold that positions them for accurate ligation. The splint acts as a mediator that eliminates deletion errors by ensuring proper alignment during the ligation reaction, thereby improving both efficiency and precision simultaneously
2Length of moving object
If multiple polynucleotide fragment molecules are ligated to form long sequences, then the desired polynucleotide product is achieved, but the process becomes complex and yields decrease
Solution Approach 1:
The patent divides the long polynucleotide synthesis into multiple manageable steps using shorter oligonucleotide fragments (10-50 mer) that are ligated sequentially. Each fragment is designed to overlap with adjacent fragments, and nucleic acid splints are used to facilitate each ligation step. This segmentation approach allows synthesis of very long sequences (hundreds of bases) while maintaining high yield at each step, avoiding the yield loss that would occur with attempting to synthesize long sequences in a single step
3Manufacturing precision
If ligation reactions are performed to join nucleic acid fragments, then the specific sequence is formed, but alignment accuracy between 3' end and 5' end is difficult to achieve
Solution Approach 1:
The nucleic acid splint serves as an intermediary scaffold that pre-organizes the 3' end of one oligonucleotide and the 5' end of another in the correct spatial arrangement for ligation. The splint contains complementary sequences to both fragments, ensuring they align accurately at their junction points. This mediator approach achieves high alignment accuracy without requiring complex reaction systems or multiple purification steps
Solution Approach 2:
The splint-mediated ligation method performs preliminary alignment of the oligonucleotide fragments before the actual ligation reaction occurs. By hybridizing the splint to both fragments in advance, the system pre-positionsthe 3' and 5' ends in the correct orientation and proximity, eliminating the need for complex real-time alignment mechanisms during ligation
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 significantly enhances the ligation efficiency, enabling the synthesis of longer polynucleotides with higher yields and facilitating purification, suitable for applications in genetic engineering and pharmaceuticals.
Implementation Method 1
the nucleic acid splints are substantially complementary to the polynucleotide molecule
Implementation Method 2
each sequence of the two or more polynucleotide fragment molecules when assembled constitutes the specific sequence
Implementation Method 3
ligating the two or more polynucleotide fragment molecules in the presence of two or more nucleic acid splints
Implementation Method 4
ligation efficiency between the 3' end side of the nucleic acid and the 5' end side of the nucleic acid
Data Source
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AI summary
The present disclosure provides a method for producing a nucleic acid molecule. Specifically, the present disclosure achieves process shortening and a dramatic improvement in ligation efficiency by using nucleic acid splints. That is, the present disclosure provides a method for producing a polynucleotide molecule having a specific sequence, the method including: 1) providing two or more polynucleotide fragment molecules including a portion of the specific sequence; and 2) ligating the two or more polynucleotide fragment molecules in the presence of two or more nucleic acid splints, wherein each sequence of the two or more polynucleotide fragment molecules when assembled constitutes the specific sequence, the nucleic acid splints are substantially complementary to the polynucleotide molecule, and at least two of the nucleic acid splints include a sequence which is substantially free of deletions when aligned with the specific sequence.