In Situ RNA Analysis via Probe Ligation and Rolling Circle Amplification
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Solution Overview
Problem
Current methods for in situ RNA analysis, such as FISH and in situ sequencing, face limitations in multiplexing, spatial resolution, and cost-effectiveness, making it challenging to simultaneously detect and localize RNA sequences with subcellular precision.
Innovation Solution
The LISH-Lock'n'Roll method involves the hybridization of probes in a sample, followed by in situ ligation to lock a circularized probe set around an RNA target sequence. Rolling circle amplification and fluorescently labeled detector probe hybridization enable simultaneous in situ quantification and localization of RNA sequences with subcellular precision.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If FISH and in situ sequencing methods are used for RNA detection, then spatial resolution is achieved, but multiplexing capability is limited due to molecular crowding requiring 40 probes per transcript
Solution Approach 1:
The patent segments the detection system into two distinct components: (1) a template-directed ligation step that creates unique circularized probe sets specific to each RNA target, and (2) a rolling circle amplification step that generates numerous copies of these circular templates. This segmentation allows a single probe set to replace what would traditionally require 40+ probes, dramatically reducing probe density while maintaining high multiplexing capability through the combinatorial specificity of the ligation step.
Solution Approach 2:
The patent performs preliminary template-directed ligation to create unique circularized probe sets before amplification. This preliminary action establishes the specific binding identity of each detection complex through enzymatic ligation, ensuring high specificity. The subsequent rolling circle amplification then mass-produces these pre-configured templates, enabling high multiplexing without requiring proportionally high probe densities in the final detection step.
2Adaptability or versatility
If serial reprobing methods like seqFISH and MERFISH are used, then RNA localization is achieved, but technical limitations prevent moving beyond certain multiplexing thresholds
Solution Approach 1:
The patent merges two previously separate techniques into a unified workflow: (1) template-directed ligation (from LISH methodology) that provides high specificity through enzymatic joining of complementary probes, and (2) rolling circle amplification that provides signal amplification. This merging creates a robust system where the specificity of ligation combined with the amplification power of RCA enables high multiplexing thresholds while maintaining technical reliability, overcoming the limitations of serial reprobing methods.
Solution Approach 2:
The patent introduces circularized probe sets as an intermediary between the target RNA and the detection system. These circular templates serve as stable, amplifiable intermediaries that encode the specific binding information. The circular intermediaries can be massively amplified and then detected, providing a reliable bridge between the initial specific ligation event and the final detection readout, thereby enabling high multiplexing with maintained reliability.
3Loss of information
If in situ sequencing methods are used, then RNA transcript information is obtained, but the same molecular crowding and multiplexing challenges as FISH persist
Solution Approach 1:
The patent uses rolling circle amplification to create numerous copies of the circularized probe templates in situ. Each circular template contains the encoded transcript information from the ligation step. By amplifying these circular templates to generate many copies, the system retains full transcript information while reducing the need for high probe densities, as the information is copied rather than requiring multiple unique probes for each detection event.
4Productivity
If scRNA-seq based methods like Drop-seq and Slide-seq are used, then whole transcriptome profiling is achieved, but high per sample cost and computational complexity prevent widespread adoption
Solution Approach 1:
The patent employs template-directed ligation where the target RNA itself serves as the template for assembling the correct probe set. The RNA transcript guides the specific joining of complementary probes through enzymatic ligation, eliminating the need for complex external sequencing and computational reconstruction. The system is self-guiding, with the target molecule directing its own detection complex assembly, thereby reducing both computational complexity and cost while maintaining whole transcriptome profiling capability.
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
LISH-Lock'n'Roll allows for high multiplexing and subcellular resolution of RNA analysis, reducing costs and simplifying workflows compared to existing methods, while maintaining high specificity and efficiency.
Implementation Method 1
incubating the reaction mixture of step (a) under conditions that permit hybridization of the at least one ligation probe set to the target sequence present in the biological sample
Implementation Method 2
adjacent hybridization of probes in a sample is followed by in situ ligation that locks a specifically circularized probe set around an RNA target sequence
Implementation Method 3
Rolling circle amplification ('Lock'n'Roll'), followed by fluorescently labeled detector probe hybridization
Implementation Method 4
amplifying the circularized probe by strand-displacement amplification
Implementation Method 5
fluorescently labeled detector probe hybridization, enables simultaneous in situ quantification and localization of RNA sequences
Data Source
AI summary
In one aspect, compositions are provided for the simultaneous in situ quantification and localization of RNA sequences with subcellular precision are utilized in the methods. In particular, specific hybridization of at least one probe set in a sample is followed by in situ ligation, which locks specifically circularized probe set around an RNA target sequence. Rolling circle amplification followed by fluorescently labeled detector probe hybridization, enables simultaneous in situ quantification and localization of RNA sequences with subcellular precision.