CAREER: Multiplexed mRNA detection at nanometer resolution

Understanding where messenger RNAs (mRNAs), molecules that carry genetic instructions from DNA to direct protein production, are located within cells is essential for explaining processes such as brain development, cellular communication, and reproductive function. However, current imaging technologies such as fluorescence microscopy and electron microscopy, cannot simultaneously identify multiple mRNAs while also revealing the extremely small cellular structures in which they operate. Fluorescence microscopy which uses light-emitting labels to detect specific molecules, can identify biological targets but cannot clearly visualize their exact locations at extremely small scales. In contrast, electron microscopy, a technique that uses beams of electrons to generate highly detailed images of cells, can reveal fine cellular structures but has limited ability to simultaneously distinguish many different label-molecules. This project addresses this major technological gap by developing a new imaging platform that combines the strengths of both approaches utilizing DNA nanotechnology, a method that uses DNA as a programable building material to create tiny structures. These tiny structures will function as barcodes by carrying identification tags for mRNAs that can be detected by both imaging techniques, enabling scientists to better understand how the brain and hormones work together to control female fertility. Because standard fluorescence and electron microscopes are already available at many universities, this approach has the potential to make advanced molecular imaging more broadly accessible to the scientific community. In parallel, the project will support interdisciplinary STEM education through undergraduate research training, engaging students from biology, chemistry, engineering, physics, and computer science in collaborative problem-solving and hands-on nanobiotechnology research. The project aligns with the Biotechnology and Artificial Intelligence (AI) priority areas of the National Science Foundation. This project will develop a modular DNA origami–based barcoding platform for multiplexed mRNA detection using both fluorescence microscopy (FM) and electron microscopy (EM), an approach known as correlative light and electron microscopy (CLEM). The proposed work will optimize the production of DNA origami barcodes containing programmable nanocrystal patterns for EM detection and fluorescent labels for FM detection, with the ability of targeting multiple specific mRNAs. Barcode assembly and performance will be characterized using gel electrophoresis, fluorometry, and EM. The platform will then be validated in neuroendocrine cell lines expressing reproductive signaling transcripts. Optimized workflows will subsequently be applied to ultrathin tissue sections to perform high-resolution spatial mapping of multiple mRNAs in hypothalamic regions relevant to reproductive neuroendocrinology. Advanced image analysis and segmentation methods, including machine learning-assisted approaches, will be used to resolve clustered barcodes and integrate FM and EM datasets, which contributes to the AI priority area of NSF. By enabling multiplexed RNA detection with nanometer-scale spatial resolution, this project will establish a new technological framework for spatial transcriptomics and multimodal bioimaging while advancing the fields of DNA nanotechnology and molecular imaging. In addition, this platform could be broadly adapted to study a wide range of biological systems requiring high-resolution molecular mapping. The project will advance the field of Biotechnology by developing an accessible toolkit using DNA-nanobiotechnology for multiplexed molecular imaging at nanometer resolution; this is a biotechnology infrastructure development that will benefit the broader scientific community. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual meri NSF Award ID: 2542984 | Program: 01003031DB NSF RESEARCH & RELATED ACTIVIT,01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Diana Goncalves Schmidt | Institution: Kent State University, KENT, OH | Award Amount: $874,717 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2542984 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2542984.html