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

这项研究旨在阐明RNA在单细胞中编码记忆和学习的机制。尽管缺乏神经元，单细胞生物，如Spirostomum ambiguum，表现出非联想形式的学习，包括习惯化。有趣的是，这些记忆可以在生物体之间转移，越来越多的证据表明RNA是一种“记忆分子”。通过利用这些单细胞生物的简单性，该项目将在分子水平上探索行为方面，绕过细胞间相互作用的复杂性。通过开发创新的微流控时间单细胞转录组学分析，将研究非编码RNA（ncRNA）在学习和记忆存储中的作用。该项目为处于学术生涯不同阶段的各种研究生科学家和学者提供了多学科学习机会。该项目还将开发双语宣传漫画，向更广泛的受众传播复杂的科学思想，促进对研究及其影响的更深入理解。这项拟议中的研究可以验证RNA作为一种分子记忆，使生物体能够长期存储和传递信息。研究表明，RNA保存的信息可以在多细胞物种（如C。线虫、扁虫、大鼠和鱼类，挑战了纯粹的突触观点。为了考虑这种现象的潜在应用，我们必须首先证明像RNA这样的分子印迹包含可以介导行为的信息。为了破译RNA中编码的潜在信息，EAGER将：i）开发适当的微流体硬件，允许非破坏性RNA提取，ii）优化工作流程和测序方案，以创建可靠的活细胞转录组学的低输入RNA-seq方法，以及iii）联合收割机这些元素，以监测习惯化和记忆转移期间的RNA变化，物种形成和时空表达谱。该研究结果可能会极大地改变我们对记忆处理的理解，测试信息编码RNA底物的存储，修改，甚至在生命和合成系统之间转移的能力。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

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.