Hybridization Chain Reaction: In Situ Amplification for Biological Imaging - Project Summary Hybridization Chain Reaction: In Situ Amplification for Biological Imaging Life is orchestrated by programmable biomolecules – DNA, RNA, and proteins – interacting within complex bio- logical circuits. RNA in situ hybridization (RNA-ISH) methods and immunohistochemistry (IHC) methods provide biologists, drug developers, and pathologists with critical windows into the spatial organization of this circuitry, enabling imaging of RNA and protein expression in an anatomical context. While it is desirable to perform multi- plex experiments in which multiple targets are imaged quantitatively at high resolution in a single specimen, using traditional RNA-ISH and IHC methods in whole-mount vertebrate embryos and thick tissue sections, multiplex- ing is cumbersome, staining is non-quantitative, and spatial resolution is routinely compromised by diffusion of reporter molecules. With traditional DNA in situ hybridization (DNA-ISH) methods, it is not currently routine to image single-copy small genomic loci in any sample, much less in vertebrate embryos. Meanwhile, methods for imaging molecular complexes have been comparatively less explored, yet represent an important frontier for spa- tial exploration of the interactome. These multi-decade technological shortcomings are significant impediments to the study of gene regulatory networks in systems most relevant to human development and disease. In situ amplification based on the mechanism of hybridization chain reaction (HCR) draws on concepts from the new field of dynamic nucleic acid nanotechnology to provide the first unified framework for multiplex, quan- titative, high-resolution RNA and protein imaging in highly autofluorescent samples. The proposed research will build on these unique capabilities to enable multiplex, quantitative, high-resolution imaging of the interactome, and to dramatically advance the performance and versatility of the HCR imaging platform. Major goals are: Imaging the interactome using cooperative probes for logical control over HCR signal amplification: mul- tiplex, quantitative, high-resolution imaging of target complexes (protein:protein, RNA:protein, RNA:RNA, DNA:DNA, DNA:protein) with high-signal to background in highly autofluorescent samples. Next-generation performance and versatility in highly autofluorescent samples: robust 15-plex spectral imaging with 1-step HCR signal amplification for all targets simultaneously; high-fidelity single-molecule RNA, protein, and complex imaging using nonlinear HCR signal amplification; compound probes for next- generation multiplex protein imaging without conventional multiplexing tradeoffs; multiplex imaging of single- copy small genomic loci with high signal-to-background; imaging sequence repeat number to provide re- searchers with the first high-resolution spatial window into mutation instability. Realization of these goals would have a broad impact on research in the biological sciences, enabling biologists, drug developers, and pathologists to perform multiplex, quantitative, high-resolution imaging of key components of the interactome, transcriptome, proteome, and genome in an anatomical context in the samples most relevant to human development and disease.
