Hybridization Chain Reaction: Automated Ultrasensitive Multiplex RNA and Protein Imaging - Project Summary Hybridization Chain Reaction: Automated Ultrasensitive Multiplex 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 drug developers, pathologists, and biologists 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 multiplex experiments in which a panel of targets is imaged with single-molecule sensitivity at low magnification over a large field-of-view within intact tissue, the dominant automated RNA-ISH and IHC methods struggle to provide these capabilities. First, multiplexing is cumbersome, requiring serial enzyme-mediated signal amplification for each target in succession (currently limited to 2-plex for chromogenic staining of RNA targets and often requiring probe stripping for protein targets). Second, sensitivity is routinely limiting at the low magnifications essential to rapid screening of large fields-of-view; this problem is only exacerbated for short RNA targets, low-expression targets, and archival tissue sections. Third, there is no unified technology for automated imaging of RNA and protein targets. Current automated RNA-ISH approaches require a protease pretreatment step that digests proteins to enable large reagents to penetrate the sample; this digestion step comes at a high cost both because it damages the tissue morphology and because it makes it challenging or impossible to image protein targets in the same sample. Collectively, these multi-decade technology gaps are significant impediments to drug development, clinical pathology, and biological research, undermining multi-dimensional analyses of genetic 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 provide the first unified framework for simultaneous RNA and protein imaging, achieving multiple breakthroughs to enable multiplex, quantitative, high-resolution RNA and protein imaging in highly autofluorescent samples. To empower high-velocity biopharma drug development and clinical diagnostics workflows, the proposed R&D will build on the unique capabilities of HCR to: 1) engineer a novel chromogenic signal amplification cascade to enable automated ultrasensitive 4-plex RNA chromogenic in situ hybridization (RNA-CISH), IHC, and simultaneous RNA-CISH/IHC at low magnification for rapid whole-slide scanning, 2) engineer a novel enzyme-free fluorescent signal ampli- fication cascade to enable automated ultrasensitive 4-plex RNA fluorescence in situ hybridization (RNA-FISH), immunofluorescence (IF), and simultaneous RNA-FISH/IF at low magnification for rapid whole-slide scanning. These capabilities will enable drug developers, pathologists, and biologists to perform automated, ultrasensi- tive, multiplex analyses of RNAs and proteins in an anatomical context using either chromogenic or fluorescent workflows.
