RNA in situ hybridization based on the mechanism of the hybridization chain reaction (HCR) enables multiplexed, quantitative, high-resolution RNA imaging in highly autofluorescent samples, including whole-mount vertebrate embryos, thick brain slices and formalin-fixed paraffin-embedded tissue sections. Here, we extend the benefits of one-step, multiplexed, quantitative, isothermal, enzyme-free HCR signal amplification to immunohistochemistry, enabling accurate and precise protein relative quantitation with subcellular resolution in an anatomical context. Moreover, we provide a unified framework for simultaneous quantitative protein and RNA imaging with one-step HCR signal amplification performed for all target proteins and RNAs simultaneously.
The lateral flow assay format enables rapid, instrumentfree, at-home testing for SARS-CoV-2. Due to the absence of signal amplification, this simplicity comes at a cost in sensitivity. Here, we enhance sensitivity by developing an amplified lateral flow assay that incorporates isothermal, enzyme-free signal amplification based on the mechanism of hybridization chain reaction (HCR). The simplicity of the user experience is maintained using a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. To perform a test, the user loads the sample, closes the device, and reads the result by eye after 60 min. Detecting gamma-irradiated SARS-CoV-2 virions in a mixture of saliva and extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/μL using available antibodies to target the SARS-CoV-2 nucleocapsid protein. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies exhibit limits of detection of 500 copies/μL, 1000 copies/μL, 2000 copies/μL, 2000 copies/μL, and 20,000 copies/μL. By swapping out antibody probes to target different pathogens, amplified HCR lateral flow assays offer a platform for simple, rapid, and sensitive at-home testing for infectious diseases. As an alternative to viral protein detection, we further introduce an HCR lateral flow assay for viral RNA detection.
RNA in situ hybridization (RNA-ISH) based on the mechanism of hybridization chain reaction (HCR) enables multiplexed, quantitative, high-resolution RNA imaging in highly autofluorescent samples including whole-mount vertebrate embryos, thick brain slices, and formalin-fixed paraffin-embedded (FFPE) tissue sections. Here, we extend the benefits of 1-step, multiplexed, quantitative, isothermal, enzyme-free HCR signal amplification to immunohistochemistry (IHC), enabling accurate and precise protein relative quantitation with subcellular resolution in an anatomical context. Moreover, we provide a unified framework for simultaneous quantitative protein and RNA imaging with 1-step HCR signal amplification performed for all target proteins and RNAs simultaneously.
The lateral flow assay format enables rapid, instrument-free, at-home testing for SARS-CoV-2. Due to the absence of signal amplification, this simplicity comes at a cost in sensitivity. Here, we enhance sensitivity by developing an amplified lateral flow assay that incorporates isothermal, enzyme-free signal amplification based on the mechanism of hybridization chain reaction (HCR). The simplicity of the user experience is maintained using a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. To perform a test, the user loads the sample, closes the device, and reads the result by eye after 60 minutes. Detecting gamma-irradiated SARS-CoV-2 virions in a mixture of saliva and extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/μL using available antibodies to target the SARS-CoV-2 nucleocapsid protein. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies exhibit limits of detection of 500 copies/μL, 1000 copies/μL, 2000 copies/μL, 2000 copies/μL, and 20,000 copies/μL. By swapping out antibody probes to target different pathogens, amplified HCR lateral flow assays offer a platform for simple, rapid, and sensitive at-home testing for infectious disease. As an alternative to viral protein detection, we further introduce an HCR lateral flow assay for viral RNA detection.
Neurolysin is a zinc metallopeptidase involved in neuropeptide degradation and modification by cleaving a number of bioactive peptides. In vivo, neurolysin hydrolyzes the short neuropeptide neurotensin, to create inactive shorter fragments. Neurolysin is of interest as a therapeutic target since a change in neurotensin level may cause a range of disorders such as pain perception, schizophrenia, addiction, cardiovascular disorders, and Huntington and Parkinson diseases. Neurolysin has a prolate ellipsoid shape with a deep channel that runs almost the entire length of the molecule. Although other metallopeptidases show a hinge-like motion, available crystal structures of neurolysin do not reveal any significant conformational changes. Thus, elucidation of its internal dynamics and how they are affected by ligand binding can aid in further design of potent ligands that either activate or inhibit neurolysin function. For this purpose, we combine two different computational techniques, namely WExplore and Elastic Network Modeling (ENM), to examine possible ligand binding sites and their effect on large-scale motions in neurolysin. WExplore uses the weighted ensemble algorithm and generates full-atomistic conformational ensembles that can span large free energy barriers. In this study, we use WExplore to generate a network of binding sites in the neurolysin channel, and predict their relative probabilities. ENM analysis, which is a fast coarse-grained normal mode analysis that provides collective deformations of proteins, reveals the hidden hinge-like motion of neurolysin, where the two domains rotate toward each other by narrowing the channel. The differences observed during hinge-like motion in apo and ligand bound forms of neurolysin pinpoint specific binding regions that affect low-frequency motions in an allosteric manner. This combined multiscale approach helps elucidate the coupling between ligand binding, hinge motion and neurolysin catalytic activity.
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