Multiplex Proximity Assays Using Shared Hybridization Sites
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Solution Overview
Problem
Existing multiplex proximity-based assays suffer from high background noise due to random interactions of unbound proximity probes, making it difficult to accurately distinguish true positive signals from false positives.
Innovation Solution
The method employs proximity probes with shared hybridization sites, allowing for the formation of background signal between all unbound probes, and uses paired barcode sequences to differentiate between true and false positive signals by comparing the presence of matched barcode sequences in the reporter nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If proximity probes with unique hybridisation sites are used for each probe pair, then specificity of analyte detection is improved, but background noise increases due to random interactions of unbound probes
Solution Approach 1:
The patent applies universality by using shared hybridisation sites that are common across multiple probe pairs. Instead of assigning unique hybridisation sites to each probe pair, the invention uses a universal set of hybridisation sites that all probes can bind to, thereby reducing background noise while maintaining detection specificity through the barcode identification system.
Solution Approach 2:
The patent introduces barcode sequences as an intermediary identification system. Rather than relying on unique hybridisation sites for probe identification, the invention uses barcodes that are read after probe binding to identify which analyte was detected. This intermediary system allows shared hybridisation sites to be used without sacrificing detection specificity.
2Measurement precision
If separate negative control assays are performed to determine background noise, then accuracy of positive signal identification is improved, but assay complexity and time consumption increase
Solution Approach 1:
The patent merges the background determination step into the main assay by using shared hybridisation sites that generate background signal within the same reaction mixture. Instead of performing separate negative control assays, the invention incorporates background generation directly into the primary assay, thereby reducing complexity and time consumption while maintaining accuracy.
Solution Approach 2:
The assay system performs self-verification by using the same shared hybridisation sites to generate both background signal and analytical signal within the same reaction. The system automatically distinguishes between background and true positive signals through barcode identification, eliminating the need for external negative control assays.
3Adaptability or versatility
If more probe pairs are included in the multiplex assay, then detection capability for multiple analytes is improved, but background noise from random interactions increases
Solution Approach 1:
The patent enables high-plex detection by using a universal set of shared hybridisation sites that can be reused across many probe pairs. This universal approach allows numerous probe pairs to be included in the multiplex assay without proportionally increasing background noise, as all probes compete for the same hybridisation sites rather than each probe pair requiring its own unique sites.
Solution Approach 2:
The invention uses barcode sequences as copyable identification tags that can be repeatedly used across different probe pairs. Instead of creating unique hybridisation site sequences for each probe pair, the system copies and reuses barcode identifiers, allowing scalable multiplexing without increasing background noise from unique sequence interactions.
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 simplifies the assay by eliminating the need for a separate negative control and provides a more accurate determination of background noise, enhancing the specificity and reliability of multiplex protein detection.
Implementation Method 1
the nucleic acid domain of each proximity probe comprises an ID sequence and at least a first hybridisation sequence, wherein the ID sequence of each proximity probe is different; and wherein: in each proximity probe pair, the first proximity probe and the second proximity probe comprise paired hybridisation sequences, such that upon binding of the first and second proximity probe to their analyte, the respective paired hybridisation sequences of the first and second proximity probes hybridise to each other or to a common splint oligonucleotide
Implementation Method 2
subjecting the duplex to an extension and/or ligation reaction to generate an extension and/or ligation product which comprises the ID sequence of the first proximity probe and the ID sequence of the second proximity probe
Implementation Method 3
subjecting the duplex to an extension and/or ligation reaction to generate an extension and/or ligation product
Data Source
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AI summary
The present invention provides a method for detecting a plurality of analytes in a sample, comprising performing a multiplex proximity-based detection assay. The assay utilises pairs of proximity probes with shared hybridisation sites (i.e. hybridisation sites which are shared between different proximity probe pairs). Also provided is a product comprising a plurality of proximity probe pairs with shared hybridisation sites, which may be used in the method disclosed herein.