Closed Nucleic Acid Structures via Stem-Loop Ligation
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Solution Overview
Problem
Current methods for generating closed nucleic acid structures are inefficient, often resulting in low yields, high side product formation, and chimeric products, making them time-consuming and complex for sequencing and diagnostic applications.
Innovation Solution
The methods involve denaturing a target nucleic acid, annealing primers with specific segments, extending them with a polymerase, and then ligating adaptors with stem-loop structures to form closed nucleic acid structures, using techniques such as PCR and exonuclease activity to create defined ends and seals nicks, thereby forming efficient and high-yield closed structures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional ligation methods are used to form closed nucleic acid structures, then the structures can be generated, but the process is complex and time-consuming with low efficiency
Solution Approach 1:
The patent applies preliminary action by pre-forming stem-loop structures on the nucleic acid targets before ligation. The 5' ends are prepared with stem-loop configurations that facilitate subsequent ligation, eliminating the need for complex in-situ structure formation during the ligation step itself. This pre-preparation simplifies the overall process and improves efficiency.
Solution Approach 2:
The patent uses stem-loop structures as intermediary elements that mediate the ligation process. These stem-loops serve as recognition and binding elements that facilitate the ligation reaction, acting as a bridge between the 5' and 3' ends of the nucleic acid targets. This intermediary mechanism simplifies the ligation process compared to direct end-to-end joining.
2Reliability
If traditional synthesis methods are used, then closed nucleic acid structures can be formed, but side product formation and chimeric products increase
Solution Approach 1:
The patent applies local quality by creating specific stem-loop structures at the 5' ends of the nucleic acid targets. These localized structural features ensure that ligation occurs at specific, defined locations rather than randomly throughout the molecule. This spatial specificity prevents misligation events that would generate chimeric products and side products, thereby improving the purity of the final closed structures.
Solution Approach 2:
By pre-forming the stem-loop structures before ligation, the patent ensures that the nucleic acid targets are properly configured for specific, accurate ligation. This preliminary structuring prevents misfolding and incorrect pairing during the ligation step, reducing the formation of side products and chimeric molecules.
3Quantity of substance
If conventional methods are used to generate closed nucleic acid structures, then the structures can be produced, but yields are low
Solution Approach 1:
The stem-loop structures act as intermediaries that facilitate efficient ligation by providing stable recognition sites and proper spatial orientation. This intermediary mechanism increases the efficiency of the ligation reaction, leading to higher yields of correctly formed closed nucleic acid structures compared to direct ligation methods.
Solution Approach 2:
The patent utilizes parameter changes by forming secondary structures (stem-loops) that alter the physical and chemical properties of the nucleic acid ends. These structural changes improve the reactivity and compatibility of the ends with the ligation enzyme, thereby increasing ligation efficiency and overall yield.
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
These methods simplify and expedite the generation of closed nucleic acid structures, reducing side products and chimerism, leading to higher yields and improved efficiency for sequencing and diagnostic applications.
Implementation Method 1
extending them with a polymerase
Implementation Method 2
using techniques such as PCR and exonuclease activity to create defined ends
Implementation Method 3
seals nicks, thereby forming efficient and high-yield closed structures
Data Source
AI summary
The invention provides compositions and methods for making closed nucleic acid structures in which one or both strands are continuous. The closed nucleic acid structures can be used as sequencing templates among other applications.