Tools4Cells: EAGER: A Molecular Pursuit for the Engram: Microfluidic temporal transcriptomics for single cell learning

This research aims to elucidate the mechanisms by which RNA encodes memory and learning in single cells. Despite their lack of neurons, single-celled organisms, such as Spirostomum ambiguum, demonstrate non-associative forms of learning, including habituation. Intriguingly, these memories can be transferred between organisms, with accumulating evidence suggesting RNA as a 'memory molecule'. By leveraging the simplicity of these single- celled organisms, the project will explore behavioral aspects at the molecular level, bypassing the complexities of intercellular interactions. Through the development of innovative microfluidic temporal single-cell transcriptomic assays, the role of non-coding RNA (ncRNA) in learning and memory storage will be investigated. The project offers a multidisciplinary learning opportunity for a diverse range of graduate student scientists and scholars at different stages in their academic careers. The project will also develop bilingual outreach comics to disseminate complex scientific ideas to a broader audience, fostering a deeper understanding of the research and its implications. The proposed research could validate RNA as a molecular memory, enabling long-term information storage and transfer in organisms. Studies have shown that RNA holds information that can transfer memories between organisms in multicellular species like C. elegans, Aplysia, flatworms, rats, and fish, challenging a purely synaptic view. To consider potential applications of the phenomena, we must first prove that a molecular engram such as RNA contains information that can mediate behavior. To decipher the potential information encoded within RNA, this EAGER will: i) develop appropriate microfluidic hardware that allows for non-destructive RNA extraction, ii) optimize the workflow and sequencing protocol to create a low-input RNA-seq approach for reliable live cell transcriptomics, and iii) combine these elements to monitor RNA changes, speciation, and spatiotemporal expression profiles during habituation and memory transfer. The results could significantly change our understanding of memory processing, testing the capacity of information-encoded RNA substrates to be stored, modified, and even transferred between living and synthetic systems. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.