Excimer Forming Fluorophores for Proximal Phosphorylation Detection
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Solution Overview
Problem
Current methods for detecting phosphorylated proteins, such as Pro-Q Diamond, cannot provide information on the spatial arrangement of phosphorylated sites, which is crucial for understanding protein activation status and disease detection, particularly in diseases like cancer where specific kinase activation is involved.
Innovation Solution
A turn-on dual emission fluorescent sensor based on an excimer forming compound that detects proximally phosphorylated sites by emitting a bathochromic shift when two or more excimer forming fluorophores associate, indicating the presence of at least two phosphorylated sites, allowing for the detection and quantification of phosphorylated proteins and pyrophosphates in various samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescent phospho-protein stains (e.g., Pro-Q Diamond) are used, then total phosphorylation levels can be determined efficiently, but spatial arrangement information of phosphorylated sites cannot be obtained
Solution Approach 1:
The invention divides the detection capability into two distinct fluorescent probes: one that detects total phosphorylation and another that specifically detects proximal phosphorylation sites. This segmentation allows simultaneous acquisition of both total phosphorylation levels and spatial arrangement information without interference between detection modes
Solution Approach 2:
The patent introduces an excimer-forming fluorophore as an intermediary component that specifically binds to proximal phosphorylated residues. This intermediary enables indirect detection of spatial arrangement by converting the spatial proximity information into a measurable excimer fluorescence signal, thereby recovering the lost spatial information
2Loss of information
If excimer forming fluorophores are used to detect proximal phosphorylation, then spatial arrangement information is obtained, but detection sensitivity and selectivity are reduced compared to conventional stains
Solution Approach 1:
The invention merges the advantages of conventional fluorescent stains with excimer-forming probes by combining high quantum yield fluorophores with excimer-forming moieties. This hybrid approach maintains the sensitivity and brightness of conventional stains while adding the spatial discrimination capability of excimer formation, thereby improving both detection precision and information content
Solution Approach 2:
The patent optimizes multiple parameters of the excimer-forming fluorophore including molecular structure, excimer formation efficiency, quantum yield, and binding affinity to phosphorylated residues. By carefully adjusting these parameters, the detection sensitivity is enhanced while preserving the spatial arrangement information, resolving the contradiction between sensitivity and information loss
3Loss of information
If multiple phosphorylation sites are detected simultaneously, then comprehensive protein activation status is obtained, but interpretation of spatial arrangement becomes complex
Solution Approach 1:
The invention employs distinct fluorescent emission characteristics (different wavelengths or excimer/monomer ratios) to encode spatial arrangement information. By using color or emission ratio changes rather than multiple separate signals, the system simplifies data interpretation while still providing comprehensive information about multiple phosphorylation sites and their spatial relationships
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
This approach enables selective and sensitive detection of proximally phosphorylated sites, providing valuable information on protein activation status and potential diagnostic tools for diseases, with applications in aqueous solutions, polyacrylamide gels, and cell culture samples.
Implementation Method 1
When two or more of the excimer forming fluorophores overlap or otherwise associate, a bathochromic shift in emission occurs, thereby increasing fluorescence intensity of the excimer-state fluorophore
Data Source
AI summary
The present application is directed to excimer forming compounds of Formula I: W—V—[Y]n wherein W is an excimer forming fluorophore, V is a linker moiety, Y is a metal ion coordinating moiety and n is 1, 2 or 3. In particular, the application is directed to excimer forming compounds for the detection of proximally phosphorylated sites including those found on polypeptides, proteins, pyrophosphate and RNA, for example in aqueous solution, polyacrylamide gels blotting membranes, solid-support assays and in cell culture samples.