Single-Stranded Splint Strands for Low-Concentration Library Circularization
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Solution Overview
Problem
Existing nucleic acid library preparation methods face inefficiencies in circularizing linear molecules, particularly at low concentrations, which can hinder downstream amplification and sequencing processes.
Innovation Solution
The use of nucleic acid single-stranded splint strands that hybridize to linear library molecules, forming a library-splint complex with a nick, which is then ligated to create covalently closed circular molecules, facilitating efficient downstream amplification and sequencing workflows.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional circularization methods are used, then the process is simple, but efficiency is poor especially at low concentrations
Solution Approach 1:
The patent introduces a single-stranded splint strand as an intermediary molecule that mediates the circularization process. The splint strand hybridizes to complementary sequences on the linear library molecule, bringing the ends together to facilitate ligation. This intermediary approach enables efficient circularization even at low library molecule concentrations where conventional direct ligation methods fail.
Solution Approach 2:
The circularization process is segmented into distinct functional regions on the splint strand: a first region that hybridizes to one end of the library molecule and a second region that hybridizes to the other end. This segmentation allows the splint strand to effectively bridge the two ends of the linear molecule, enabling circularization through controlled, stepwise interaction rather than random collision.
2Productivity
If splint strands are used to improve circularization efficiency, then efficiency improves, but reannealing of splint strands may occur
Solution Approach 1:
The splint strand is designed with non-uniform local properties: the first and second regions have sequences specifically complementary to the library molecule ends, while the middle region has sequences that prevent self-complementarity. This local quality differentiation ensures that the splint strand binds to the library molecule rather than reannealing to itself, maintaining process reliability.
Solution Approach 2:
The patent optimizes parameters including the length and sequence composition of different splint strand regions, the hybridization temperature, and the molar ratios of splint strand to library molecule. These parameter changes are tuned to favor splint-strand-library complex formation over splint strand self-annealing, thereby maintaining high efficiency while preventing reannealing artifacts.
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 high-efficiency circularization of library molecules, even at low concentrations, improving the reliability and efficiency of nucleic acid amplification and sequencing processes.
Implementation Method 1
The single-stranded splint strand comprises a first region that hybridizes with a sequence on one end of the linear single stranded library molecule, and a second region that hybridizes with a sequence on the other end of the linear single stranded library molecule
Implementation Method 2
The nick can be ligated to form covalently closed circular molecules
Data Source
AI summary
The present disclosure provides compositions comprising nucleic acid single-stranded splint strands, including kits, and methods that employ the single-stranded splint strands. The single-stranded splint strands can hybridize to portions of linear library molecules to form circularized library-splint complexes having a nick, where the nick can be ligated to form covalently closed circular molecules which can be subjected to downstream amplification and sequencing workflows.