Designing a Hybrid Chain Reaction Probe for Multiplex Transcripts Assay with High-Level Imaging

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Hybrid chain reaction (HCR) is an enzyme-free, isothermal amplification method widely used in fluorescent in situ hybridization (FISH).
• Existing HCR methods suffer from time-consuming protocols and low signal efficiency, limiting their utility in transcriptomic assays.
• There is a need for improved HCR strategies to enhance signal amplification and imaging quality for multiplex applications.

Purpose of the Study:

• To develop novel orthogonal HCR hairpin-pair (hp) probes for efficient signal amplification in multiplex assays.
• To enhance the efficiency and imaging capabilities of HCR-based multiplex FISH assays.
• To create a simplified and more effective tool for spatial cell type and transcriptome analysis.

Main Methods:

• Developed nine orthogonal HCR hairpin-pair (hp) probes.
• Coupled fluorescent molecules to HCR hairpins via disulfide bonds for easy chemical cleavage, simplifying the workflow.
• Combined HCR with in situ rolling circle amplification (ISRCA) to create the ISRCA-HCR technique.

Main Results:

• The developed HCR probes enabled efficient signal amplification for multiplex assays.
• The ISRCA-HCR technique achieved a significant 17-fold signal amplification.
• ISRCA-HCR demonstrated high-level imaging capability, suitable for spatial cell type assays.

Conclusions:

• The study presents a novel ISRCA-HCR method that overcomes limitations of existing HCR techniques.
• The developed HCR probes and ISRCA-HCR strategy provide accurate, high-level signal amplification for multiplex FISH imaging.
• This offers a powerful research tool for advanced transcriptome and spatial cell type analysis.