Rhodamine Dye Reactive Groups for Nucleic Acid Labeling Efficiency
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Solution Overview
Problem
Current fluorescent dyes used for labeling nucleic acids and other molecules face challenges such as reduced incorporation efficiency by polymerases, cumulative loss of product in synthetic oligonucleotides, and impractical synthesis of longer nucleic acid lengths, necessitating improved labeling methods and linker arm selections.
Innovation Solution
Development of novel rhodamine dyes with specific structural features, including various reactive groups and energy transfer capabilities, for efficient labeling and fluorescence energy transfer systems, enabling effective covalent bonding or binding pair interactions with target molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional fluorescent dyes are used for labeling nucleic acids, then labeling can be achieved, but incorporation efficiency by polymerases is reduced
Solution Approach 1:
The patent modifies the chemical structure of fluorescent dyes by changing parameters such as introducing reactive groups (isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, haloacetamide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, phosphoramidite) and adjusting molecular properties to enhance polymerase incorporation efficiency while maintaining fluorescence labeling capability
Solution Approach 2:
The patent employs reactive groups as intermediary functional moieties that facilitate covalent bonding between the fluorescent dye and target molecules (nucleic acids, proteins, peptides). These reactive groups act as mediators that enable efficient incorporation by polymerases while maintaining the fluorescent properties needed for detection
2Reliability
If conventional dyes are used in synthetic oligonucleotides, then labeling is achieved, but cumulative loss of product occurs
Solution Approach 1:
The patent employs transient reactive groups that can be easily introduced and removed or stabilized during the synthesis process. The reactive groups are designed to be temporary during synthesis but become stable in the final product, preventing cumulative loss. Alternatively, the patent uses disposable-like strategies where the reactive groups are optimized for single-use efficiency in the synthesis context
Solution Approach 2:
The patent introduces protective groups and stabilized reactive moieties beforehand during dye synthesis to prevent degradation and loss during subsequent oligonucleotide synthesis steps. The reactive groups are pre-optimized to resist cumulative loss through proper chemical stabilization while maintaining labeling efficiency
3Reliability
If conventional labeling methods are used, then short nucleic acids can be labeled, but synthesis of longer nucleic acids becomes impractical
Solution Approach 1:
The patent segments the labeling process into modular steps where reactive groups are introduced at specific positions along the nucleic acid sequence. This allows incremental labeling of longer nucleic acids by dividing the synthesis into manageable segments, each with optimized reactive groups, rather than attempting to label entire long molecules in a single step
Solution Approach 2:
The patent employs dynamic reactive groups that can adapt their reactivity based on the synthesis context. The reactive groups are designed to be highly reactive during incorporation but stable in the final product, allowing efficient labeling of longer nucleic acids without compromising fidelity. The reactivity is dynamically adjusted through chemical modification
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
Enhances labeling efficiency, reduces product loss, and allows for longer nucleic acid synthesis, improving the spectral profiles and energy transfer pair selection for real-time detection and amplification assays.
Implementation Method 1
fluorescent dyes useful for labeling nucleic acids and other molecules
Implementation Method 2
reactive group Z that reacts with the target molecule to form a covalent bond between reactive group Z and the target molecule
Implementation Method 3
fluorescence energy transfer systems, enabling effective covalent bonding or binding pair interactions with target molecules
Data Source
AI summary
Provided are methods for labeling target molecules, such as nucleic acids, with fluorescent dye compounds having the formulaOne method embodiment includes contacting reactive group Z of the fluorescent dye compound with the target molecule such that reactive group Z reacts with the target molecule to form a covalent bond between the group and the target molecule. Another method embodiment includes contacting a fluorescent dye compound that further includes a first member of a binding pair, with a target molecule that includes a second member of the binding pair. Also provided are target molecules labeled with the fluorescent dye compounds.