Tricyclic Polymethine Dyes for Spectrally Resolved Nucleic Acid Sequencing
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Solution Overview
Problem
Existing fluorescent dyes for nucleic acid sequencing face challenges in achieving spectrally distinguishable absorption and emission spectra, compatibility with reagent chemistries, and stability under high excitation conditions, which complicates multiplex detection and increases sequencing errors and costs.
Innovation Solution
Development of tricyclic polymethine dyes with tailored Stokes shifts, improved fluorescence intensity, and enhanced chemical and thermal stability, allowing for covalent attachment to nucleotides for use in nucleic acid sequencing applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple fluorescent dyes are used together for multiplex detection, then the number of reaction vessels is reduced and throughput is increased, but it becomes difficult to find dye compounds with substantially resolved absorption and emission spectra
Solution Approach 1:
The patent applies parameter changes by systematically modifying the chemical structure of fluorescent dyes, specifically adjusting the polymethine chain length and cyclic group configurations to achieve distinct Stokes shifts. This allows multiple dyes to be differentiated by their unique emission spectra when excited at the same wavelength, resolving the spectral overlap issue while maintaining high throughput multiplex detection
Solution Approach 2:
The invention uses composite fluorescent dye structures combining cyclic groups with polymethine chains of varying lengths. These composite molecular structures exhibit different Stokes shifts due to their unique electronic properties, enabling spectral discrimination of multiple dyes simultaneously excited by a single light source, thus achieving both high productivity and spectral resolution
2Illumination intensity
If high power light sources like lasers are used for excitation, then fluorescence signals are enhanced, but the dye must have sufficient photo-stability to withstand such excitation
Solution Approach 1:
The patent employs composite fluorescent dye structures with cyclic groups attached to polymethine chains. This composite architecture provides enhanced photo-stability compared to linear polymethine dyes, allowing the dyes to withstand high power laser excitation without rapid photodegradation, thus maintaining reliable fluorescence signals over extended sequencing runs
Solution Approach 2:
The cyclic group structures in the fluorescent dyes provide inherent structural stability and resistance to photodegradation before exposure to excitation light. This pre-engineered structural robustness cushions against the damaging effects of high power laser excitation, extending the operational lifetime of the dyes during multiplex sequencing
3Productivity
If fluorescent dyes are used in water-based biological buffers at higher temperatures, then sequencing reactions proceed efficiently, but the fluorescence intensities of most organic dyes are significantly lower under such conditions
Solution Approach 1:
The patent applies parameter changes by modifying the molecular structure of fluorescent dyes to include cyclic groups that enhance quantum yield and fluorescence intensity. These structural modifications compensate for the quenching effects of water-based buffers and elevated temperatures, maintaining strong fluorescence signals under physiological sequencing conditions without sacrificing reaction efficiency
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 tricyclic polymethine dyes provide improved fluorescence signals, reduced sequencing errors, and decreased reagent usage, enhancing the efficiency and accuracy of nucleic acid sequencing processes.
Implementation Method 1
tricyclic polymethine dyes with tailored Stokes shifts, improved fluorescence intensity
Implementation Method 2
dyes with different Stokes shifts. The Stokes shift is the difference between the absorption maximum wavelength and the emission maximum wavelength
Data Source
AI summary
The present application relates to dyes containing a tricyclic polymethine core and their uses as fluorescent labels. These dyes may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications.