Reversible DNA-Tagged Affinity Probes for Multiplex Biomarker Profiling
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Solution Overview
Problem
Existing methods for molecular profiling, particularly in cell biology and clinical diagnostics, face limitations in simultaneously analyzing multiple biomarkers due to the inability to uniquely match fluorescent probes with corresponding biomarkers, leading to restricted multiplexing capabilities and inadequate understanding of disease processes.
Innovation Solution
A hybrid IF/FISH method is developed, utilizing unique DNA tags to encode biomarkers in the first step and QD-DNA or DNA-FL probes in the second step, enabling multiplexed detection and quantification of analytes through reversible conjugation of affinity molecules to label moieties, allowing for extensive multiplexing and sequential staining cycles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional biomedical techniques are used for molecular profiling, then the methods are simple to operate, but the number of biomarkers that can be analyzed simultaneously is limited
Solution Approach 1:
The patent introduces DNA tags as intermediary molecules that bridge affinity molecules (antibodies) and fluorescent probes. Each affinity molecule is conjugated to a unique DNA tag, and complementary DNA-conjugated fluorescent probes specifically hybridize to these tags, enabling unique matching and multiplexed detection of multiple biomarkers simultaneously
Solution Approach 2:
The detection system is segmented into distinct functional modules: affinity molecules for biomarker binding, DNA tags for encoding identity, and fluorescent probes for detection. This segmentation allows independent optimization of each component and enables combinatorial multiplexing by varying the DNA tag sequences
2Reliability
If direct QD-affinity molecule conjugation is used for multiplexed staining, then the staining process is quick and direct, but the ability to uniquely match each probe with corresponding biomarker is limited
Solution Approach 1:
DNA tags serve as information-carrying intermediaries that provide unique molecular barcodes for each affinity molecule-probe pair. The complementary base pairing between DNA tags and DNA probes ensures specific and reliable matching, enabling multiplexed detection with high confidence in probe-biomarker correspondence
Solution Approach 2:
The system varies the nucleotide sequence parameters of DNA tags to encode different affinity molecules. By changing the DNA sequence while maintaining the same affinity molecule structure, the system creates distinguishable probes that can be detected with high specificity
3Productivity
If conventional immunohistochemical staining is performed, then the staining conditions are optimized for each antibody, but sequential staining cycles cannot be performed without removing previous stains
Solution Approach 1:
The patent employs reversible conjugation between affinity molecules and DNA-conjugated fluorescent probes through hybridization-dehybridization cycles. After detection, probes can be removed by heating or chemical treatment, allowing the same sample to undergo multiple staining cycles with different probe sets, thereby increasing productivity
Solution Approach 2:
The conjugation between affinity molecules and probes is made dynamic and reversible through temperature-controlled hybridization and dehybridization. This dynamic system allows easy removal of probes after detection, facilitating sequential staining cycles without permanent modification of the sample
Data Source
AI summary
Provided herein are compositions and methods for identifying or quantitating one or more analytes in sample. The composition can comprise an affinity molecule reversibly conjugated to a label moiety via a double-stranded nucleic acid linker or via an adaptor molecule. The affinity molecule and the label moiety can be linked to different strands of the double-stranded nucleic acid linker. Compositions can be used in any biological assays for detection, identification and/or quantification of target molecules or analytes, including multiplex staining for molecular profiling of individual cells or cellular populations. For example, the compositions can be adapted for use in immunofluorescence, fluorescence in situ hybridization, immunohistochemistry, western blot, and the like.