Two-Probe RNA Proximity Ligation for Multiplexed Single-Cell Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for analyzing mRNA in single cells are limited by the number of detectable fluorophores, laborious and costly processes, and the need for complex probe designs, leading to inefficient multiplexed experiments and variability in gene expression measurements, especially in complex samples like tumors.
Innovation Solution
The SNAIL-RCA method uses Splint Nucleotide Assisted Intramolecular Ligation followed by Rolling Circle Amplification, employing two oligonucleotides (SPO and PO) that hybridize to adjacent mRNA regions, allowing for specific circularization and high-throughput detection of multiple transcripts in single cells, compatible with flow and mass cytometry.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple short fluorescently labelled nucleotide probes are used for single molecule RNA detection, then detection sensitivity is improved, but probe synthesis complexity and cost increase
Solution Approach 1:
The patent combines two separate probe functions into a single oligonucleotide probe that contains both the target-binding region and the ligation site. This merged probe design eliminates the need for synthesizing multiple separate probes while maintaining detection sensitivity through the proximity ligation mechanism.
Solution Approach 2:
The oligonucleotide probe is designed with multi-functionality, serving both as the target-binding probe and as the substrate for proximity ligation. The probe contains universal components (binding region, ligation site, fluorophore) that can be applied across different RNA detection applications, reducing synthesis complexity.
2Measurement precision
If complex four-probe systems with intermediate hybridization sequences are used, then RNA detection capability is improved, but experimental design labor and time increase
Solution Approach 1:
The patent extracts and eliminates the intermediate hybridization sequence component from the detection system. By using a direct proximity ligation approach where the probe binds directly to the target RNA and undergoes ligation, the method removes the time-consuming intermediate steps while maintaining detection capability.
Solution Approach 2:
The oligonucleotide probe is pre-designed with all necessary functional elements (target-binding region, ligation site, fluorophore) already in place. This preliminary preparation of a self-sufficient probe eliminates the need for sequential hybridization steps and intermediate sequence design, significantly reducing experimental design time.
3Measurement precision
If conventional microscopy with multiple fluorophores is used for spatial distribution analysis, then spatial resolution is improved, but the number of simultaneously detectable targets is limited
Solution Approach 1:
The patent employs fluorophores with different emission wavelengths (colors) that can be detected by conventional microscopy. Each oligonucleotide probe is labeled with a specific fluorophore, enabling multiplexed detection of multiple RNA targets simultaneously while maintaining spatial resolution through optical detection.
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 method enables cost-effective, high-sensitivity detection of multiple nucleic acids and proteins in single cells, with high specificity and signal-to-noise ratios, suitable for analyzing complex samples like biopsies and blood samples, and can detect genomic variations and fusion events.
Implementation Method 1
two oligonucleotides (SPO and PO) that hybridize to adjacent mRNA regions
Implementation Method 2
The ligated circle is formed by ligation of the 5' phosphate to the 3' hydroxyl group of the padlock oligonucleotide
Implementation Method 3
The circle is amplified by rolling circle amplification using phi29 DNA polymerase
Data Source
AI summary
SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second. The methods require only two primers for amplification, and may further include a detection primer.