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Programmable in situ amplification for multiplexed imaging of mRNA expression

• Niles Pierce, Caltech

Abstract

Each cell in a multi-cellular organism contains the same genome, yet the regulatory circuits encoded within this genome implement a developmental program yielding significant spatial heterogeneity and complexity. In situ hybridization methods are an essential tool for elucidating developmental and pathological processes, enabling imaging of mRNA expression in a morphological context from sub-cellular to organismal length scales. Due to variability between specimens, accurate mapping of spatial relationships between the regulatory loci of different genes requires multiplexed experiments in which multiple mRNAs are imaged in a single biological sample. With current in situ hybridization approaches, it is challenging to simultaneously detect the expression of multiple target mRNAs within intact vertebrate embryos – a significant impediment to the study of interacting regulatory elements in systems most relevant to human development and disease. To address this challenge, we have engineered programmable molecular amplifiers based on the mechanism of hybridization chain reaction (HCR). Using this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background and sharp signal localization.