Two-Probe RNA Visualization for Multiplexed Single-Cell Detection
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Solution Overview
Problem
Existing methods for analyzing mRNA in single cells are limited by the number of detectable fluorophores, laborious and costly procedures, and the need for complex probe designs, leading to inefficient multiplexed experiments and variability in gene expression measurements.
Innovation Solution
The SNAIL-RCA method uses a two-probe system comprising a Splint Primer Oligonucleotide (SPO) and Padlock Oligonucleotide (PO) to hybridize adjacent regions of mRNA, followed by rolling circle amplification, enabling specific and efficient detection of multiple transcripts in single cells.
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 the number of probes that can be simultaneously detected by conventional microscope is limited
Solution Approach 1:
The patent introduces a secondary detection system using padlock probes and rolling circle amplification as an intermediary step. Instead of directly detecting multiple fluorophores on mRNA, the method first hybridizes padlock probes to the mRNA, then uses rolling circle amplification to generate circular DNA templates that can be detected by fluorescently labeled oligonucleotides. This intermediary amplification step enables detection of multiple transcripts simultaneously with higher sensitivity than direct fluorophore detection.
Solution Approach 2:
The patent changes the detection parameter from direct fluorophore counting to amplification-based signal generation. By using rolling circle amplification, a single mRNA molecule can generate multiple copies of the circular DNA template, each capable of binding detection oligonucleotides with fluorophores. This parameter change from 1:1 detection to 1:n amplification significantly improves detection sensitivity and enables multiplexed detection beyond the limit of conventional fluorophore detection.
2Measurement precision
If complex four-probe system with intermediate hybridization sequences is used for proximity-based RNA detection, then detection capability is improved, but probe design complexity and labor requirement increase
Solution Approach 1:
The patent extracts and eliminates the intermediate hybridization sequence component from the four-probe system. Instead of using separate capture probes and intermediate sequences, the method uses simple padlock probes that directly hybridize to the target mRNA with adjacent sequences. The padlock probe design requires only two hybridization regions flanking the target site, removing the need for complex intermediate sequences and reducing probe design complexity while maintaining detection capability.
Solution Approach 2:
The patent creates a universal two-probe system where padlock probes can be designed for any target mRNA using a standardized format. The padlock probe structure with 5' and 3' ends flanking the target site, followed by universal ligation and rolling circle amplification steps, provides a multi-functional platform that can detect any RNA target without requiring custom intermediate sequences for each gene. This universal approach significantly reduces probe design complexity compared to gene-specific four-probe systems.
3Productivity
If bulk transcriptome measurements are used, then measurement efficiency is improved, but single-cell gene expression variability is lost
Solution Approach 1:
The patent segments the bulk transcriptome measurement into single-cell measurements. By applying the padlock probe and rolling circle amplification method to individual cells, the technique enables measurement of gene expression in each cell separately while maintaining high throughput. The segmentation allows preservation of single-cell gene expression variability while still achieving efficient measurement through automated processing and high-capacity detection platforms.
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 allows for high-throughput, cost-effective detection of multiple nucleic acids and proteins in single cells, with high specificity and sensitivity, compatible with cytometry and microscopy, and capable of analyzing complex cell populations.
Implementation Method 1
hybridize adjacent regions of mRNA
Implementation Method 2
followed by rolling circle amplification
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.