Cleavable Linkers for Stable Immobilized Transposome Tagmentation
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Solution Overview
Problem
Current nucleic acid library preparation methods using immobilized transposomes face issues with stability and efficiency, leading to reduced read enrichment and off-target capture, particularly when using biotin-streptavidin interactions for hybridization capture.
Innovation Solution
The use of modified transposome complexes with cleavable linkers that attach to solid supports, allowing for improved stability and reduced off-target capture, enhancing the efficiency of nucleic acid library production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If biotin-streptavidin interactions are used for immobilizing transposomes, then the library preparation is simplified and faster, but the stability of the support-bound transposome complexes deteriorates and read enrichment is reduced
Solution Approach 1:
The patent changes the chemical parameters of the linker by incorporating cleavable bonds (photolabile, redox-sensitive, enzymatically cleavable) that allow controlled release under specific conditions. This resolves the contradiction by maintaining strong binding during library prep while enabling stable complex formation during sequencing, thus improving both productivity and reliability through parameter modification.
Solution Approach 2:
The patent introduces dynamic linkers that can change their binding state based on environmental conditions (light, redox potential, enzyme presence). The linker transitions from a bound state during library preparation to a released state during sequencing, allowing the system to adapt its properties to different process requirements and simultaneously achieve high productivity and reliability.
2Ease of operation
If transposomes are immobilized on solid support, then hands-on time and reagent requirements are reduced, but off-target capture increases and manufacturing precision deteriorates
Solution Approach 1:
The patent applies local quality by designing linkers with specific cleavable bonds at particular positions (5' or 3' end of transposon) to control where and when release occurs. This localized control ensures that fragment size uniformity is maintained during the critical tagmentation step while still benefiting from the ease of operation provided by immobilization.
Solution Approach 2:
The cleavable linker acts as an intermediary between the solid support and the transposome complex. It mediates the interaction by allowing controlled attachment during library prep and controlled release during sequencing, thus maintaining manufacturing precision while preserving ease of operation.
3Adaptability or versatility
If transposome complexes are removed from support during storage, then accessibility is improved, but quality and efficiency of the resulting library deteriorates
Solution Approach 1:
The patent uses dynamic linkers that remain bound during storage and library preparation, then release under specific conditions (light, redox, enzyme treatment). This dynamic behavior maintains library quality by preventing premature removal while enabling accessibility when needed through controlled release mechanisms.
Solution Approach 2:
The cleavable linker provides beforehand cushioning by being designed to remain stable under storage conditions but become labile under specific trigger conditions. This protects the transposome complex from premature removal while ensuring future accessibility when the appropriate release conditions are met.
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
The modified transposome complexes provide more stable and efficient nucleic acid library production with consistent insert sizes and reduced off-target capture, improving sequencing outcomes.
Implementation Method 1
The transposome complexes are used to fragment and tag target nucleic acids in solution to generate a sequencer-ready tagmented library
Implementation Method 2
The transposome complexes may be immobilized on a solid surface, such as through a biotin appended at the 5′ end of one of the two end sequences
Implementation Method 3
During the subsequent PCR amplification step of the protocol, biotin-streptavidin bonds are broken by thermal denaturation, thereby releasing the biotinylated tagmentation product into solution
Data Source
AI summary
The present disclosure relates to methods, compositions, and kits for treating target nucleic acids, including methods and compositions for fragmenting and tagging nucleic acid (e.g., DNA) using transposome complexes bound to a solid support.