Multiplex RNA In Situ Hybridization With Cleavable Signal Amplification
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Solution Overview
Problem
Existing RNA in situ hybridization (ISH) methods face limitations in multiplexing capability due to the small number of spectrally distinct fluorescent dyes, leading to substantial loss of nucleic acid detection sensitivity and requiring time-consuming sequential hybridization and detection steps.
Innovation Solution
A method involving multiple rounds of target probe sets, pre-amplifiers, amplifiers, and distinguishable cleavable label probes to simultaneously detect multiple target nucleic acids, allowing for higher multiplexing without significant sensitivity loss.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If sequential rounds of hybridization and detection are used to increase multiplexing, then the number of target sequences detected increases, but nucleic acid detection sensitivity and cellular morphology are substantially lost
Solution Approach 1:
The detection system is segmented into distinct functional components: target probes that bind to specific RNA sequences, pre-amplifiers that bind to target probes, amplifiers that bind to pre-amplifiers and carry cleavable fluorescent labels, and cleavage enzymes that remove labels after detection. This segmentation allows simultaneous detection of multiple targets without sequential processing, preserving sensitivity.
Solution Approach 2:
Pre-amplifiers serve as intermediary molecules between target probes and amplifiers. Each pre-amplifier contains binding sites for both target probes and amplifiers, enabling the transfer of binding information without direct interaction between target probes and amplifiers. This intermediary layer allows complex multiplexed detection while maintaining signal integrity.
2Adaptability or versatility
If sequential hybridization methods are used to detect multiple targets, then more target sequences can be imaged, but the process becomes time and labor intensive
Solution Approach 1:
Multiple detection channels are merged into a single simultaneous detection process. Different target probe sets are labeled with different fluorescent dyes that can be detected at the same time using spectral unmixing or sequential excitation/detection cycles. The cleavable label system allows all targets to be detected in one hybridization event rather than requiring sequential processing.
Solution Approach 2:
The detection process uses periodic action through controlled cleavage of fluorescent labels. After simultaneous detection of all targets, cleavage enzymes are introduced to remove labels in a controlled manner, allowing the system to reset for additional detection cycles without requiring complete removal and re-hybridization of all probes.
3Adaptability or versatility
If more fluorescent dyes are used to increase multiplexing, then more target sequences can be visualized, but the optical systems can only distinguish a limited number of spectrally distinct dyes
Solution Approach 1:
The system changes the detection parameter from relying solely on spectral distinction to using temporal and chemical parameters. Cleavable fluorescent labels allow the system to detect targets sequentially in time rather than requiring all targets to be spectrally distinct. The cleavage step resets the fluorescent signal, enabling the same optical channel to detect multiple targets at different time points, effectively increasing multiplexing capability without requiring proportionally more spectrally distinct dyes.
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 simultaneous visualization of a higher number of target sequences with improved sensitivity and reduced time and labor, overcoming the limitations of traditional ISH methods.
Implementation Method 1
each target probe set comprises a pair of target probes that specifically hybridize to a target nucleic acid
Implementation Method 2
each pre-amplifier comprises binding sites for the pair of target probes of one of the target probe sets and a plurality of binding sites for an amplifier
Implementation Method 3
Fluorescent RNA ISH utilizes fluorescent dyes and fluorescent microscopes for RNA labeling and detection, respectively
Implementation Method 4
the labels in each first subset of label probes are distinguishable between the first subsets of label probes and wherein the labels are cleavable
Data Source
AI summary
The invention relates to methods of multiplex detection of a plurality of target nucleic acids by contacting a sample comprising a cell with target probe sets that specifically hybridize to target nucleic acids, with pre-amplifiers or pre-pre-amplifiers specific for each target probe set, with amplifiers specific for the pre-amplifiers, and with label probes specific for the amplifiers, resulting in specific labeling of multiple target nucleic acids. The invention also relates to samples, slides and kits relating to detection of multiple target nucleic acids.