Research Project
10158198
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 auto?uorescent 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 signi?- 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 ampli?cation 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 auto?uorescent samples including whole-mount vertebrate embryos, thick brain slices, and FFPE tissue sections: 1) straightforward multiplexing with 1-step quantitative signal ampli?cation 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 bene?ts of 1-step multiplexed quantitative enzyme-free HCR signal ampli?cation to IHC, and to develop the ?rst in situ ampli?cation product for simultaneous RNA-ISH/IHC, performing 1-step HCR signal ampli?cation for targets RNAs and proteins simultaneously. During Phase I, we will engineer molecular components to enable highly multiplexed signal ampli?cation, and establish a uni?ed 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.
