Fluorescent Dye Linkers for Quenching-Resistant 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, leading to reduced sensitivity and efficiency in 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 maintain dye separation and optimize molecular quenching, thereby enhancing nucleic acid processing and detection efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent dyes are used for nucleic acid sequencing, then detection sensitivity is improved, but dye brightness is attenuated due to quenching phenomena
Solution Approach 1:
A linker molecule is introduced as an intermediary between the fluorescent dye and the nucleic acid substrate. The linker serves as a spatial mediator that separates the dye from quenching sources (biological materials, other dyes, solvent) while maintaining functional coupling for detection. This physical separation reduces quenching effects and preserves dye brightness, directly resolving the contradiction between detection sensitivity and brightness attenuation.
2Productivity
If dyes are placed close to substrates for efficient labeling, then labeling efficiency is improved, but quenching by proximity to other dyes increases
Solution Approach 1:
The linker is designed with specific local structural properties including rigid aromatic rings and appropriate length to create a localized separation zone between the dye and substrate. This local structural quality allows the dye to be positioned close enough for efficient energy transfer and labeling while maintaining sufficient distance from quenching sources, thereby preserving brightness while maintaining labeling efficiency.
3Illumination intensity
If conventional linkers are used to separate dye from quencher, then quenching is reduced, but solvent-dependence and polydispersity issues arise
Solution Approach 1:
The linker is designed as a composite molecular structure combining rigid aromatic rings with flexible alkyl chains and hydrophilic groups. This composite architecture provides both structural rigidity for fixed geometry (reducing polydispersity) and solvent compatibility through hydrophilic interactions. The linker maintains consistent performance across different solvent conditions, eliminating solvent-dependence while preserving the brightness enhancement effect.
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 proposed linker-dye system improves nucleic acid sequencing sensitivity and efficiency by minimizing quenching effects, allowing for clearer signal detection and more accurate sequencing results.
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... A general solution to many types of quenching requires physical separation of the dye from the quencher moiety
Implementation Method 3
the linker comprises (i) one or more water soluble groups... The linker is configured to establish a functional length of at least about 0.5 nanometers (nm) between the fluorescent dye and the substrate
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.