Friday, April 25, 2025
4/25/2025
Hybridization Chain Reaction: Highly Multiplexed Quantitative RNA and Protein Imaging - Project Summary Hybridization Chain Reaction: Highly Multiplexed Quantitative RNA and Protein Imaging Encoded in the genome of each organism, biological circuits direct development, maintain integrity in the face of attacks, control responses to environmental stimuli, and sometimes malfunction to cause disease. RNA in situ hybridization (RNA-ISH) and immunohistochemistry (IHC) methods provide biologists, drug developers, and pathologists with critical windows into the spatial organization of this circuitry, enabling imaging of mRNA and protein expression in an anatomical context. While it is desirable to perform multiplexed experiments in which a panel of targets is imaged quantitatively at high resolution in a single specimen, using traditional RNA-ISH and IHC methods in highly autofluorescent samples including whole-mount vertebrate embryos and FFPE tissue sections, multiplexing is cumbersome or impractical, spatial resolution is frequently compromised by diffusion of reporter molecules, and staining is non-quantitative. These multi-decade technological shortcomings are signifi- cant impediments to biological research as well as to advancement in drug development and pathology assays, preventing high-dimensional quantitative analyses of developmental and disease-relevant regulatory networks in an anatomical context. To overcome these challenges, in situ amplification based on the mechanism of hybridization chain reaction (HCR) draws on concepts from the emerging discipline of dynamic nucleic acid nanotechnology to achieve four RNA-ISH breakthroughs in highly autofluorescent samples including whole-mount vertebrate embryos, thick brain slices, and FFPE tissue sections: 1) straightforward multiplexing with 1-step quantitative signal amplification for up to 5 target mRNAs simultaneously; 2) analog mRNA relative quantitation with subcellular resolution in an anatomical context; 3) digital mRNA absolute quantitation with single-molecule resolution in an anatomical context; 4) automatic background suppression throughout the protocol, dramatically enhancing performance and ease-of-use. The proposed research will build on the unique capabilities of HCR to enable next-generation levels of multiplexing for RNA-ISH, to extend the benefits of 1-step multiplexed quantitative enzyme-free HCR signal amplification to IHC, and to develop the first in situ amplification product for simultaneous RNA-ISH/IHC, performing 1-step HCR signal amplification for targets RNAs and proteins simultaneously. During Phase I, we will engineer molecular components to enable highly multiplexed signal amplification, and establish a unified framework for RNA and protein imaging. During Phase II, we will develop and commercialize robust technologies for highly multiplexed RNA-ISH/IHC in key sample types critical for academic research, drug development, and/or clinical diagnostics. These new HCR products will enable biologists, drug developers, and pathologists to perform previously impossible quantitative analyses on large panels of mRNAs and proteins at high resolution with full anatomical context.
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