Oligonucleotide Probes for Single Molecule Spatial Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
There is an unmet need for improved methods of detecting single molecules, such as proteins or peptides, by using the spatial positions of the molecules.
Innovation Solution
The method involves interrogating a sample of dilute proteins using iterative proximity ligation (IPL) or split-and-pool labeling with oligonucleotide probes to determine positional information, allowing for the identification of molecular neighborhoods and the detection of single molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If conventional DNA barcoding is used to label and track molecules, then molecule identification capability is improved, but spatial position detection precision deteriorates
Solution Approach 1:
The oligonucleotide probe is segmented into multiple functional regions: a functional group for attachment to the molecule, a primer site for DNA synthesis, a unique barcode for identification, and a 5' cleavage half-site or 3' cleavage half-site for proximity ligation. This segmentation allows each region to perform its specific function independently, enabling simultaneous molecule identification and spatial position detection without interference between these functions.
Solution Approach 2:
The oligonucleotide probe acts as an intermediary between the molecule of interest and the detection system. By attaching the probe to the molecule and using its structured design with barcode and cleavage sites, it mediates the connection between molecular identity and spatial information, allowing both properties to be detected through the same probe structure.
2Measurement precision
If iterative proximity ligation is performed to detect spatial positions, then spatial resolution is improved, but detection complexity increases
Solution Approach 1:
The iterative proximity ligation process employs periodic cycles of ligation and cleavage. In each iteration, oligonucleotide tags on nearby molecules are ligated together, then a catalyst is added to cleave them apart. This periodic repetition of ligation-cleavage cycles progressively builds up linkage information between spatially proximal molecules, achieving high spatial resolution through multiple incremental steps rather than a single complex operation.
Solution Approach 2:
The catalyst added in each iteration temporarily modifies the oligonucleotide tags by adding nucleotides, but then the cleavage step removes these modifications and restores the original tags. This discarding and recovering process allows the same oligonucleotide tags to participate in multiple ligation cycles without permanent modification, reducing the need for continuously adding new reagents and simplifying the overall detection system.
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 the detection and identification of single molecules at the nanometer or micrometer scale, providing detailed spatial information and allowing for the identification of proteins or peptides in a sample.
Implementation Method 1
ligating oligonucleotide tags with a 5' cleavage half-site to oligonucleotide tags with a 3' cleavage half-site which are held in proximity, thereby generating one or more barcode pairs
Implementation Method 2
extending the primer in one of the oligonucleotide tags of the one or more barcode pairs to generate duplicates of the barcode pairs
Implementation Method 3
adding a catalyst for cleavage to separate the oligonucleotide tags
Data Source
AI summary
The present disclosure provides methods for molecular neighborhood detection of molecules, such as by iterative proximity ligation or split-and-pool methods for obtaining positional information.