Padlock Probe Rolling Circle Amplification for RNA Localization
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Solution Overview
Problem
Current methods for detecting RNA expression levels and localization in tissues face challenges such as diffusion and loss of reaction products, lack of sensitivity and specificity, and limitations in multiplex detection due to spectral overlap and background interference, failing to provide accurate in-situ detection and spatial information.
Innovation Solution
A method using V-type and C-type probe sets, combined with padlock probes and rolling circle amplification, allows for simultaneous detection of multiple target nucleic acids in cells, employing a ligase to form circular polynucleotides and amplify signals for precise localization and quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional in-situ hybridization is used to detect RNA, then in-situ detection can be achieved, but the reaction products diffuse and break away making accurate localization difficult
Solution Approach 1:
The patent combines multiple probes (target probe, padlock probe, detection probe) into an integrated detection system where the padlock probe circularizes upon binding to the target, creating a stable structural complex that prevents diffusion and loss of reaction products while maintaining precise localization capability
Solution Approach 2:
The patent employs a composite probe system consisting of multiple nucleic acid probes with different functions (target recognition, circularization, signal detection) that work together to achieve both stable product formation and accurate localization, overcoming the limitations of single-probe systems
2Measurement precision
If single-molecule fluorescence in-situ hybridization with multiple fluorophores is used, then detection sensitivity is improved, but spectral overlap limits the number of RNAs that can be detected simultaneously
Solution Approach 1:
The patent assigns different detection probes with distinct spectral properties to different target RNAs within the same tissue section, allowing each target to be detected with optimal sensitivity while enabling multiplex detection by spatially resolving different fluorescence signals
Solution Approach 2:
The patent transitions from detecting multiple targets in different spectral dimensions (which causes overlap) to detecting multiple targets in the same spectral dimension through sequential or spatially resolved imaging, adding a temporal or spatial dimension to resolve the spectral overlap problem
3Quantity of substance
If single-cell sequencing technology is used to analyze cell population heterogeneity, then quantitative RNA expression information is obtained, but accurate cell type source and location information is lost
Solution Approach 1:
The patent uses tissue section imaging as an intermediary that bridges single-cell sequencing and spatial context, allowing RNA expression quantification to be performed on isolated cells while the spatial location information is preserved through coordinate mapping and overlay with the tissue section image
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 accurate, sensitive, and specific detection of RNA expression levels and localization, improving hybridization efficiency and reducing non-specific detection, capable of detecting multiple targets in a single sample with high signal-to-noise ratio.
Implementation Method 1
the padlock probe is capable of hybridizing or annealing to the first complementary sequence of the first probe and the second complementary sequence of the second probe to form a circular polynucleotide with a nick
Implementation Method 2
contacting the product of step (b) with a ligase under a condition that allows the ligase to ligate a nucleic acid nick
Implementation Method 3
performing rolling circle amplification of the product of step (b) by using an amplification enzyme under a condition that allows the amplification
Implementation Method 4
contacting the product of the previous step with the detection probe under a condition that allows hybridization or annealing, and detecting a signal from the detection probe bound to the product
Data Source
AI summary
The present invention relates to a method for testing the presence or level of one or more target nucleic acids in a sample, and further relates to a probe set and a kit comprising one or more probe sets.