Polymethine Fluorescent Dyes for Multiplex Sequencing
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Solution Overview
Problem
Current fluorescent markers for nucleic acid sequencing face challenges in achieving high power saturation, spectral distinguishability, and compatibility with reagents, leading to reduced fluorescence intensity and increased sequencing errors, especially in multiplex detection methods.
Innovation Solution
Development of novel polymethine compounds with improved fluorescence properties, including specific structural modifications such as indole N-phenyl moieties, sulfonic acid groups, and linker attachments, which enhance photostability and allow for efficient conjugation with nucleotides, enabling better excitation efficiency and spectral resolution.
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 spectral resolution and excitation efficiency become difficult to maintain
Solution Approach 1:
The patent modifies molecular parameters of fluorescent dyes, specifically the polymethine chain length and indole N-substitution patterns, to systematically tune absorption and emission spectra. This allows optimization of spectral spacing between multiple dyes while maintaining efficient excitation, resolving the contradiction between multiplex capability and spectral resolution.
Solution Approach 2:
The patent introduces specific local structural modifications at the indole N-position with different substituents (phenyl, alkyl, heteroaryl groups) to create distinct spectral signatures for each dye in the multiplex set. This local differentiation enables clear spectral resolution while maintaining overall multiplex functionality.
2Illumination intensity
If high power light sources are used for excitation, then fluorescence signal strength is improved, but photo-stability of the dye is compromised
Solution Approach 1:
The patent creates composite fluorescent structures combining polymethine dyes with indole cores and specific N-substituents, where the indole moiety provides enhanced photostability while the polymethine chain maintains strong fluorescence. This composite structure allows high signal strength with improved resistance to photodegradation.
Solution Approach 2:
The patent converts the potential harm of high power excitation (which causes dye degradation) into a benefit by designing dyes with extended conjugation and stable indole cores that actually benefit from high excitation power. The robust molecular structure transforms the previously harmful high-energy excitation into an advantageous condition for achieving strong signals without excessive degradation.
3Measurement precision
If fluorescent dyes are optimized for spectral properties, then multiplex detection capability is improved, but compatibility with reagent chemistries is reduced
Solution Approach 1:
The patent designs polymethine dyes with functional groups and charge distributions that provide universal compatibility across different reagent chemistries while maintaining optimized spectral properties. The indole core and polymethine structure serve multiple functions: spectral tuning, chemical stability, and broad reagent compatibility, allowing the same dye structure to function effectively in various sequencing and detection protocols.
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 new polymethine compounds achieve higher power saturation levels, improve fluorescence intensity, and reduce sequencing errors, facilitating more accurate and efficient nucleic acid sequencing, particularly in high-throughput methods and multiplex detection applications.
Implementation Method 1
new polymethine compounds and their use as fluorescent markers
Implementation Method 2
absorption bands of the dyes which could be useable for this are usually widely separated
Data Source
AI summary
The present disclosure relates to new compounds and their use as fluorescent labels. The compounds may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications.