Semi-Rigid Fluorescent Linkers for Low-Quenching Sequencing
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Solution Overview
Problem
Existing nucleic acid sequencing technologies face challenges in maintaining dye brightness due to quenching phenomena, such as quenching by biological materials, proximity to other dyes, and solvent effects, which affect the sensitivity and efficiency of nucleic acid detection and sequencing.
Innovation Solution
The use of fluorescent labeling reagents comprising a fluorescent dye connected to a linker with specific structural features, including water-soluble groups and ring systems, to optimize molecular quenching and enhance nucleic acid processing and detection, with the linker configured to establish a functional length between the dye and the substrate.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent dyes are used for nucleic acid labeling, then detection sensitivity is improved, but quenching effects reduce dye brightness
Solution Approach 1:
A linker molecule is introduced as an intermediary between the fluorescent dye and the nucleic acid substrate. The linker has specific structural features including water-soluble groups and rigid ring systems that prevent direct contact between the dye and quenching agents (biological materials, solvent, or other dyes), thereby reducing quenching effects while maintaining fluorescent signal for detection
Solution Approach 2:
The harmful quenching interaction is extracted and separated from the dye-substrate complex by introducing the linker as a spatial barrier. The linker physically removes the quencher from the proximity of the fluorescent dye, allowing the dye to maintain its brightness while still enabling detection of the nucleic acid
2Productivity
If dyes with high brightness are used, then detection efficiency is improved, but quenching by proximity to other dyes occurs
Solution Approach 1:
The linker acts as a mediator that separates the fluorescent dye from other dye molecules in the solution. By incorporating rigid ring systems and water-soluble groups, the linker creates sufficient spatial distance between adjacent dyes to prevent dipole-dipole quenching and other proximity-based harmful interactions, while still allowing efficient detection
3Illumination intensity
If physical separation of dye from quencher is implemented, then quenching is reduced, but existing solutions have limitations in ease of use and polydispersity
Solution Approach 1:
The linker is designed with specific parameter changes in its molecular structure, including the incorporation of water-soluble groups (such as hydrophilic moieties) and rigid ring systems with defined geometries. These parameter changes ensure the linker provides consistent quenching protection across different conditions while maintaining solubility and reducing polydispersity issues that plague existing separation solutions
Solution Approach 2:
The linker functions as a composite molecular structure combining multiple functional elements: water-soluble groups for biological compatibility and dispersion, rigid ring systems for structural integrity and quenching separation, and functional groups for covalent attachment to both the dye and the nucleic acid substrate. This composite approach overcomes the limitations of simpler separation methods
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 solution improves the efficiency and sensitivity of nucleic acid sequencing by reducing quenching effects, allowing for optimized fluorescent labeling and detection, thereby enhancing the accuracy and reliability of genetic analysis.
Implementation Method 1
The detection, quantification and sequencing of cells and biological molecules may be important for molecular biology and medical applications... Nucleic acid sequencing may comprise the use of fluorescently labeled moieties... The fluorescent labeling reagent coupled to the substrate is configured to emit a fluorescent signal
Implementation Method 2
Dye brightness may be attenuated by quenching phenomena, including quenching by biological materials, quenching by proximity to other dyes, and quenching by solvent... Molecular quenching mechanisms can include photoinduced electron transfer, photoinduced hole transfer, Forster energy transfer, Dexter quenching
Data Source
AI summary
The present disclosure provides labeling reagents for labeling substrates such as nucleotides, proteins, antibodies, lipids, and cells. The labeling reagents provided herein may comprise fluorescent labels and semi-rigid linkers. Methods for nucleic acid sequencing using materials comprising such labeling reagents are also provided here.