PLISH RNA Detection via Holliday Junctions and Rolling Circle Amplification
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Solution Overview
Problem
Current molecular profiling techniques face challenges in multiplexed single-molecule in situ hybridization in tissues due to autofluorescent background and light scattering, requiring laborious probe hybridization and signal amplification steps, with limited RNA-detection efficiency and compatibility with intact tissue samples.
Innovation Solution
The use of proximity ligation-in situ hybridization (PLISH) with oligonucleotide probes forming Holliday-like junctions for ultrasensitive transcript detection, incorporating bridge and circle oligonucleotides for rolling circle amplification, ensuring specificity and high signal generation, compatible with automated image analysis for multiplex expression profiling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If hybridization chain reaction (HCR) is used to amplify probe signals, then detection sensitivity is improved, but the process becomes laborious due to repeated hybridization and amplification cycles
Solution Approach 1:
The patent combines probe hybridization and signal amplification into a single simultaneous reaction step, eliminating the need for repeated hybridization and amplification cycles required by HCR. The proximity ligation mechanism enables both detection and amplification to occur together, dramatically reducing processing time while maintaining high detection sensitivity.
Solution Approach 2:
The patent uses padlock probes that are pre-designed with complementary sequences that will form a circular structure upon hybridization to adjacent target RNA molecules. This preliminary design allows the amplification to occur immediately after hybridization without requiring separate amplification cycles, thus reducing overall processing time.
2Loss of time
If unamplified smISH techniques are used, then processing time is reduced, but signal strength is insufficient for tissue detection
Solution Approach 1:
The patent employs rolling circle amplification where a single circularized padlock probe template automatically generates multiple copies of the signal sequence without requiring external intervention or additional reagents. This self-amplifying mechanism provides strong signal generation in tissues while maintaining a simple, fast single-step process.
Solution Approach 2:
The patent changes the amplification mechanism from enzymatic HCR to rolling circle amplification, which operates under different biochemical parameters that are more compatible with tissue sections. This parameter change enables adequate signal strength in tissues while keeping the process simple and fast.
3Measurement precision
If multiple probe hybridization steps are performed for multiplexed detection, then RNA-detection efficiency is improved, but device complexity increases
Solution Approach 1:
The patent uses a universal padlock probe design that can detect multiple different RNA targets by simply changing the probe sequences. The same basic mechanism (hybridization, ligation, rolling circle amplification) works for all targets, enabling multiplexed detection without increasing procedural complexity. This is further enhanced by the ability to perform repeated rounds of probe hybridization to the same tissue section.
Solution Approach 2:
The patent segments the detection process into distinct molecular components (probe regions, padlock regions, barcode regions) that can be independently designed and combined. This modular segmentation allows efficient detection of multiple RNA species by simply mixing different probe sets without complicating the overall workflow.
4Measurement precision
If classical proximity ligation with RCA is used, then signal amplification is achieved, but compatibility with intact tissue samples is limited due to autofluorescent background and light scattering
Solution Approach 1:
The patent introduces a barcode sequence as an intermediary between the target RNA and the fluorescent detection system. The rolling circle amplification generates multiple copies of this barcode, which then serves as a robust platform for fluorescent labeling. This intermediary approach provides sufficient signal amplification to overcome tissue autofluorescence and light scattering issues.
Solution Approach 2:
The patent creates a composite signal structure combining the padlock probe, circularized DNA, rolled-circle amplicons, and fluorescently-labeled imager oligonucleotides. This composite approach integrates multiple functional elements that work together to provide strong, specific signal detection in challenging tissue environments despite autofluorescence and scattering.
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
PLISH enables efficient, high-throughput detection of multiple RNA species in tissues with enhanced sensitivity and specificity, providing single-molecule resolution and compatibility with intact tissue samples, overcoming previous limitations in RNA-detection efficiency and multiplexing.
Implementation Method 1
hybridization of fluorescently-labeled oligonucleotide probes, typically 24-96 per gene, to mark individual RNA molecules
Implementation Method 2
Rolling Circle Amplification (RCA) of padlock probes
Implementation Method 3
fluorescently-labeled oligonucleotide probes
Data Source
AI summary
Compositions and reagents for molecular profiling using proximity ligation-/>7 situ hybridization (PLISH) are disclosed. In particular, PLISH merges the specificity of proximity ligation, the sensitivity of tiling multiple probes for a target nucleic acid, and the high signal intensity of rolling circle amplification. The probe design capitalizes on the formation of Holliday-like junctions for optimal signal amplification. PLISH provides single molecule resolution and allows for quantitation of a virtually unlimited number of nucleic acids within individual cells. PLISH is also compatible with immunohistochemistry and archival formal-fixed, paraffin-embedded tissue samples.