Molecular Instruments, Inc

Advancing Probe Technology for Ultrasensitive Rna Imaging via Hybridization Chain Reaction - Project Summary Advancing Probe Technology for Ultrasensitive Rna Imaging via Hybridization Chain Reaction 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) Methods Provide Drug Developers, Pathologists, and Biologists With a Critical Window Into the Spatial Organization of This Circuitry, Enabling Imaging of Rna Expression in an Anatomical Context. While It Is Desirable to Perform Multiplex Experiments in Which Multiple Targets Are Imaged Quantitatively at High Resolution in a Single Specimen, Using Traditional Rna-ish Methods in Whole-mount Vertebrate Embryos and Thick Tissue Sections, Multiplexing Is Cumbersome, Staining Is Non-quantitative, and Spatial Resolution Is Routinely Compromised by Diffusion of Reporter Molecules. These Multi-decade Technological Shortcomings Have Significantly Impeded the Study of Gene Regulatory Networks in Systems Most Relevant to Human Development and Disease. to Overcome These Challenges, 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 Redefine the State-of-the- Art for Rna Fluorescence in Situ Hybridization (rna-fish), Achieving Four Breakthroughs in Highly Autofluorescent Samples Including Whole-mount Vertebrate Embryos, Thick Brain Slices, and Ffpe Tissue Sections: 1) Straight- Forward Multiplexing With 1-STEP Quantitative Signal Amplification for Up to 10 Target Mrnas Simultaneously; 2) Analog Mrna Relative Quantitation With Subcellular Resolution in an Anatomical Context; 3) Digital Mrna Abso- Lute Quantitation With Single-molecule Resolution in an Anatomical Context; 4) Automatic Background Suppression Throughout the Protocol, Dramatically Enhancing Performance and Ease-of-use. However, Automatic Background Suppression Is Achieved Using a Dual-probe Technology That Does Not Apply to Short Rna Targets (e.g., Mirnas) That Are Only Long Enough to Stably Bind a Single Probe. Moreover, for Single- Molecule Imaging of Low-abundance Mrna Targets, Where Each Target Molecule Is Resolved as a Distinct Dot, Variable Probe Hybridization Yield Due to Competing Secondary Structure Within the Target Leads to a Distribution of Dot Intensities That Can Overlap With Autofluorescence, Leading to False-negatives or False-positives. the Proposed R&D Will Address These Two Critical Technology Gaps. to Suppress Background for Imaging Short Rna Targets, We Will Develop a New Probe Architecture That Minimizes Non-specific Binding While Preserving Robust HCR Am- Plification. to Achieve High-fidelity Single-molecule Imaging Across All Classes of Rna Targets, We Will Develop an Automated Probe Design Pipeline That Combines Physics-based Simulations With Bioinformatics to Generate Probe Sequences With Minimal Off-target Binding and High-yield Hybridization to Cognate Rna Targets. These Advances Will Enable Biologists, Drug Developers, and Pathologists to Perform Ultrasensitive Imaging of All Classes of Target Rnas With Anatomical Context in the Samples Most Relevant to Human Development and Disease.