The complex organization of chromatin (DNA with its associated nuclear proteins) is closely tied to the regulation of gene expression, and evidence points to a central role for chromatin deregulation in tumor development. A common analysis of chromatin regulatory protein localization is the chromatin immunoprecipitation (ChIP) assay, which involves crosslinking of proteins to DNA, followed by chromatin fragmentation, and then antibody mediated immunoprecipitation of the DNA fragments. Subsequent quantification (quantitative PCR or Next Generation Sequencing) is usually performed. This assay is a key tool for both industry and academic research studying cancer-related epigenetics The challenge is that the formaldehyde crosslinks render the chromatin resistant to mechanical (i.e. acoustic sonication) lysis, making current fragmentation techniques inefficient and time-consuming. Furthermore, these techniques result in high sample-to-sample variability and require high-power, low- throughput, and expensive sonication devices. Our customer discovery interviews have highlighted these pain points; there is an urgent need for a higher quality and higher throughput method of chromatin fragmentation before assays such as ChIP can be incorporated into cancer diagnostics applications. Triangle Biotechnology recently developed a unique cavitation enhancing reagent (RapidShear) that substantially improves the efficiency of acoustic fragmentation of genomic DNA. However, this reagent formulation shows little to no improvement for chromatin fragmentation from fixed cells. To address the aforementioned sample preparation challenges in the epigenetics space, we are developing a new reagent, MegaShear, a more aggressive formulation for acoustic cavitation enhancement designed specifically for chromatin processing. Preliminary studies indicated that MegaShear facilitates a substantial improvement in chromatin fragmentation throughput, consistency, and yield. Validation and optimization of this technology will alleviate a critical bottleneck in the ChIP workflow. MegaShear will enable a substantial leap in high quality sample preparation throughput desired by large companies, as well as an increase in accessibility by enabling the use of low-cost sonicators, desired by small academic labs. With this SBIR, we will demonstrate proof of concept for MegaShear, validating the ability to produce high quality chromatin with high consistency, reducing capital equipment cost, and substantially increasing throughput. Success of this project will enable a novel commercial solution for addressing market pain points in chromatin analysis.
Public Health Relevance Statement: RELEVANCE TO PUBLIC HEALTH Growing evidence points to a central role for chromatin deregulation in tumor development, and assays such as the chromatin immunoprecipitation (ChIP) assay are key tools for both industry and academic research in cancer-related epigenetics. However, issues with consistency and throughput have severely limited the utility and adoption of ChIP for personalized medicine applications. To solve this problem, Triangle Biotechnology has developed MegaShear, a cavitation enhancement reagent with applications for biological sample dispersion and chromatin fragmentation. MegaShear can significantly decrease sample processing time and substantially increase throughput, eliminating the barriers in chromatin analysis and enabling robust, high-throughput biological sample processing for ChIP and other epigenetic assays.
Project Terms: Acoustics; Address; Adoption; Antibodies; Bathing; Biological; Biological Assay; Biotechnology; cancer biomarkers; Cancer Diagnostics; Capital; Cell Line; cell type; Cells; Chromatin; chromatin immunoprecipitation; companion diagnostics; Complex; Consumption; cost; crosslink; Cytolysis; Data; design; Development; Devices; Diagnostic; Digestion; DNA; Enzymes; Epigenetic Process; Equipment; experimental study; Formaldehyde; Formulation; Gene Expression; Gene Expression Regulation; Genes; Genetic; genetic regulatory protein; Genetic Transcription; Genomic DNA; Gold; histone modification; Histones; Immunoglobulin G; Immunoprecipitation; improved; Industry; Industry Standard; instrument; interest; Interview; Laboratories; Lead; Letters; Malignant Neoplasms; Manufacturer Name; Mechanics; Mediating; Methods; Modification; next generation sequencing; novel; Nuclear Proteins; Pain; Pathway interactions; personalized medicine; Post-Translational Protein Processing; Preparation; Problem Solving; Procedures; Process; protein crosslink; Proteins; Protocols documentation; Public Health; Reagent; Research; research and development; research study; Resistance; Role; Sampling; Signal Transduction; Small Business Innovation Research Grant; Sonication; success; System; Techniques; Technology; Therapeutic; Time; tool; Translations; tumor; Ultrasonics; Validation; Variant; Water; Work
