Probe Ligation for Precise Analyte Proximity Detection
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Solution Overview
Problem
Current methods for analyzing the proximity of analytes within a sample are limited in their ability to accurately detect and quantify the proximity of specific analytes, particularly in complex biological samples such as tissue samples, without causing significant disruption or alteration.
Innovation Solution
A method involving the use of probes with specific binding sites and barcodes, followed by ligation and amplification to generate amplicons, which are then detected using detection probes, allowing for the determination of analyte proximity through the detection of barcode complements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods are used to analyze analyte proximity in tissue samples, then the sample can be analyzed, but the methods lack precision in detecting and quantifying analyte proximity
Solution Approach 1:
The detection system is segmented into distinct functional components: probes with specific binding sites for different analytes, ligation enzymes for joining probe ends, and detection systems for reading the joined products. This segmentation allows each component to be optimized independently for its specific function, improving overall measurement precision without requiring a monolithic complex system
Solution Approach 2:
The patent introduces circular oligonucleotide intermediaries that serve as mediators between the analytes and the detection system. These circular oligonucleotides are formed by ligating probe ends together when probes bind to adjacent analytes, creating a stable intermediate structure that can be specifically detected and quantified, thereby enhancing proximity detection precision
2Reliability
If probes are used to detect analyte proximity, then detection accuracy improves, but the procedure becomes more complex
Solution Approach 1:
The patent combines multiple functions into the probe structure itself: the probe contains both binding sites for specific analyte recognition and ends that can be ligated together. This merging of recognition and signaling functions into a single probe molecule simplifies the overall procedure by reducing the number of separate reagents and steps needed while maintaining high detection reliability
Solution Approach 2:
The probe structure is designed to be self-assembling and self-signaling. When probes bind to adjacent analytes, their ends automatically come into proximity and can be ligated together without additional manipulation, creating a self-evident signal of analyte proximity. This self-service mechanism reduces procedural complexity while ensuring reliable detection
3Measurement precision
If circular oligonucleotides are formed through ligation, then analyte proximity can be precisely determined, but the process time increases
Solution Approach 1:
The probe structures are pre-designed and pre-positioned in the sample before the ligation step. Multiple probes simultaneously bind to their target analytes in parallel, and only after this preliminary binding phase is complete is the ligation enzyme added. This preliminary action allows the system to prepare all necessary probe-analyte complexes simultaneously, reducing the overall process time while maintaining precise spatial relationship measurements
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
Enables precise detection of the proximity of analytes in biological samples, including nucleic acids and proteins, by forming circular oligonucleotides and amplifying them to generate detectable signals, thereby providing accurate information on their spatial relationships.
Implementation Method 1
a first probe and a second probe, wherein the first probe comprises: (i) a first binding site configured to couple to a first analyte at a first portion of the first analyte; (ii) a second binding site configured to couple to the first analyte at a second portion of the first analyte
Implementation Method 2
ligating the first end and the second end to form a circular oligonucleotide
Implementation Method 3
amplifying the circular oligonucleotide to generate an amplicon, wherein the amplicon comprises a complement of the barcode
Implementation Method 4
detecting the complement of the barcode or a derivative thereof using a plurality of detection probes
Data Source
AI summary
Provided herein are methods and systems for identifying a proximity of analytes in a sample. The method may comprise contacting one or more analytes with one or more probes. The proximity of the one or more analytes to each other may cause a ligation event between the one or more probes. An amplification reaction may be performed comprising one or more copies of a barcode or derivative thereof. The one or more copies of the barcode or derivative thereof may be detected to identify a proximity between the one or more analytes.