Asymmetric Splint Oligonucleotides for ssDNA Cyclization
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Solution Overview
Problem
Existing library construction technologies for DNA nanoball sequencing platforms face low cyclization efficiency of single-stranded nucleic acid molecules due to improper choice of splint nucleic acid molecules, leading to linear complexes and imbalanced base separation, affecting DNA quantification and SNP determination accuracy.
Innovation Solution
A splint nucleic acid molecule with asymmetric complementary regions of different lengths is used to facilitate two-stage annealing, avoiding intermolecular ligation and enhancing self-cyclization efficiency by using a thermostable ligase like Taq DNA ligase.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a conventional splint nucleic acid molecule is used for cyclization, then the cyclization reaction can proceed, but the cyclization efficiency is low and linear complexes are formed
Solution Approach 1:
The patent applies asymmetry by designing the splint nucleic acid molecule with two complementary regions of different lengths (first complementary region and second complementary region). This asymmetric structure prevents the splint from forming linear complexes with multiple single-stranded nucleic acid molecules, as the mismatched lengths prevent proper alignment for intermolecular ligation. Meanwhile, the asymmetric design still allows intramolecular cyclization to proceed effectively.
2Measurement precision
If the splint nucleic acid molecule allows annealing, then cyclization can occur, but base separation occurs affecting accuracy
Solution Approach 1:
The asymmetric length design of the two complementary regions prevents the splint from mediating intermolecular ligation between different single-stranded nucleic acid molecules. This eliminates the formation of linear complexes that would cause base separation during sequencing, thereby improving DNA quantification accuracy and SNP determination reliability.
3Productivity
If symmetric complementary regions are used in the splint, then annealing is simplified, but intermolecular ligation occurs reducing self-cyclization efficiency
Solution Approach 1:
The patent employs asymmetric complementary region lengths to prevent intermolecular ligation while maintaining effective intramolecular cyclization. The first complementary region and second complementary region have different lengths, which sterically and thermodynamically prevent the splint from bridging two different DNA molecules, thereby enhancing self-cyclization efficiency.
Solution Approach 2:
The splint nucleic acid molecule is designed with different local properties in its two complementary regions. The first complementary region has a different length than the second complementary region, creating local asymmetry that specifically prevents intermolecular interactions while allowing intramolecular cyclization to proceed efficiently.
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 improves the intramolecular self-ligation efficiency of single-stranded nucleic acid molecules, reducing base separation rates to 0.5% or below, ensuring high accuracy in DNA quantification and SNP determination.
Implementation Method 1
the 5′ terminal fragment is adapted to form a first complementary region between the 5′ terminal fragment and a 5′ terminal of the single-stranded nucleic acid molecule, the 3′ terminal fragment is adapted to form a second complementary region between the 3′ terminal fragment and a 3′ terminal of the single-stranded nucleic acid molecule
Data Source
AI summary
Provided in the present invention are a splint nucleic acid molecule for cyclizing a single-stranded nucleic acid molecule and an application therefor. The splint nucleic acid molecule is composed of a 5′ terminal fragment and a 3′ terminal fragment, the 5′ terminal fragment being adapted to forming a first complementary region with a 5′ terminal of the single-stranded nucleic acid molecule, and the 3′ terminal fragment being suited to forming a second complementary region with a 3′ terminal of the single-stranded nucleic acid molecule, the length of the first complementary region and the second complementary region being different.