A novel platform for quantification of acute neuronal transcriptional responses

用于量化急性神经元转录反应的新平台

基本信息

批准号：
10600925
负责人：
Zu-Wen Sun
金额：
$ 32.65万
依托单位：
EPICYPHER, INC.
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-15 至 2024-09-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10600925
关键词：
Acute Address Antibodies Bar Codes Benchmarking Binding Biological Assay Brain Cells ChIP-seq Chimeric Proteins Chromatin Clinical Research Code Complex DNA DNA-Directed RNA Polymerase Data Development Elements Enhancers Epitopes Estradiol Exhibits Feasibility Studies Fentanyl G-substrate Genes Genetic Transcription Genomics Goals Gold Histones Hormones Human Cell Line Hyperactivity In Situ Learning Ligation MCF7 cell Maps Masks Measurement Measures Memory Messenger RNA Modeling Neurobiology Neurons Neurosciences Neurosciences Research Noise Nucleosomes Opioid Pharmacotherapy Phase Phosphorylation Phosphorylation Site Play Poly(A)+ RNA Polymerase Population Positioning Attribute Process Proteins Protocols documentation RNA RNA Polymerase II RNA chemical synthesis Research Rodent Model Role Run-On Assays Sampling Series Services Signal Transduction Site Stimulus Substance Use Disorder Technology Testing Time Tissues Tn5 transposase Transcription Elongation Transcription Initiation Untranslated RNA Work base brain tissue commercialization cost cross reactivity data quality driving force drug development drug discovery experimental study extracellular feeding improved innovation mRNA sequencing novel nucleotide analog opioid exposure phase 1 study relating to nervous system research and development response substance use success

项目摘要

PROJECT SUMMARY Acute transcriptional responses underlie essential neurobiological processes from development to learning and memory, and have been hypothesized to be a driving force behind substance use disorders. However, current assays to measure acute changes in transcription lack the sensitivity and throughput to power detailed, mechanistic studies of the immediate transcriptional response to neuronal stimuli. Widely used mRNA- seq assays measure transcription by quantifying bulk levels of stable, polyadenylated RNA, which mask early transcriptional responses. mRNA-seq also fails to capture non-protein coding RNAs, such as enhancer RNAs (eRNAs), which are correlated with enhancer activity and play essential roles in enhancer function in neurons. Assays that measure newly synthesized ‘nascent RNAs’ are capable of measuring early changes in transcription, including eRNAs. However, current assays to measure nascent RNA synthesis require complex workflows and exhibit poor assay sensitivity, limiting their use for neuroscience research. Rather than measuring nascent RNAs, direct measurement of RNA polymerase provides a powerful approach to study transcription that overcomes these limitations. RNA polymerase II (RNAPII) is the polymerase that transcribes protein coding mRNAs and non-coding RNAs, including eRNAs, and contains a carboxyl-terminal domain (CTD) that is differentially phosphorylated to promote transcription initiation and elongation. Mapping the genomic position of RNAPII and its phosphoforms provides a unique opportunity to directly measure transcriptional dynamics, but current assays to map RNAPII (e.g. ChIP-seq) lack the sensitivity to accomplish this. Here, EpiCypher is developing CUTANA-RNAPII, an ultra-sensitive RNAPII mapping assay that directly quantifies global transcriptional dynamics in response to extracellular stimuli for neuroscience research and drug development. The innovation of our proposal is the development of a modified CUT&Tag workflow that targets various RNAPII CTD phosphorylation sites to measure early changes in transcription initiation and elongation. The goal of this Phase I feasibility study is to demonstrate the sensitivity of CUTANA-RNAPII assays to measure early transcriptional responses in neurons. In Aim 1, we will develop the CUTANA-RNAPII workflow and benchmark our assay against the current gold-standard nascent RNA assay (PRO-seq assay) to measure transcriptional dynamics in cells in response to hormone treatment. In Aim 2, we will work with Dr. Zoe McElligott to demonstrate the ability of CUTANA-RNAPII to measure early transcriptional dynamics in a neuroscience model of opioid exposure. Following success in Phase I, in Phase II we will develop robust CUTANA-RNAPII beta kits and automated assay services that are optimized for brain tissue using low sample inputs, including single cells. This work will be key for deploying CUTANA-RNAPII to study highly heterogeneous brain samples for breakthrough neuroscience and substance use research. This low-cost assay platform will revolutionize how transcriptional dynamics can be studied in complex tissues for clinical research and drug development research.

项目概要急性转录反应是从发育到发育的重要神经生物学过程的基础学习和记忆，并已被发展为药物滥用障碍背后的驱动力。然而，目前测量转录急剧变化的测定缺乏灵敏度和通量对广泛使用的 mRNA 的即时转录反应进行详细的机制研究。 seq 检测通过量化稳定的多聚腺苷酸 RNA 的大量水平来测量转录，这些 RNA 会掩盖早期 mRNA-seq 也无法捕获非蛋白质编码 RNA，例如增强子 RNA。（eRNA），与增强子活性相关，并在神经元增强子功能中发挥重要作用。测量新合成的“新生 RNA”的测定能够测量转录的早期变化，然而，目前测量新生 RNA 合成的方法需要复杂的工作流程和表现出较差的敏感性测定，限制了它们在神经科学研究中的应用，而不是测量新生的 RNA。直接测量 RNA 聚合酶为研究转录提供了一种强大的方法，克服了这些限制是 RNA 聚合酶 II (RNAPII) 是转录蛋白质编码 mRNA 的聚合酶。非编码 RNA，包括 eRNA，并含有一个羧基末端结构域 (CTD)，该结构域与磷酸化以促进转录起始和延伸。绘制 RNAPII 的基因组位置。其磷酸形式提供了直接测量转录动态的独特机会，但目前的检测绘制 RNAPII 图谱（例如 ChIP-seq）缺乏实现这一目标的灵敏度。 EpiCypher 正在开发 CUTANA-RNAPII，这是一种超灵敏的 RNAPII 作图分析方法，可直接量化响应细胞外刺激的全局转录动态，用于神经科学研究和药物我们提案的创新之处在于开发了针对目标的修改后的 CUT&Tag 工作流程。各种 RNAPII CTD 磷酸化位点，用于测量转录起始和延伸的早期变化。该 I 期可行性研究的目标是证明 CUTANA-RNAPII 测定法测量的敏感性在目标 1 中，我们将开发 CUTANA-RNAPII 工作流程和将我们的测定与当前黄金标准新生 RNA 测定（PRO-seq 测定）进行基准测试以测量在目标 2 中，我们将与 Zoe McElligott 博士合作研究细胞中对激素治疗的转录动态。证明 CUTANA-RNAPII 测量神经科学中早期转录动态的能力继第一阶段的成功之后，我们将在第二阶段开发强大的 CUTANA-RNAPII。使用低样本输入针对脑组织进行优化的 beta 试剂盒和自动检测服务，包括这项工作将是部署 CUTANA-RNAPII 研究高度异质性大脑样本的关键。该低成本检测平台将彻底改变神经科学和物质使用研究的方法。可以在复杂组织中研究转录动力学，用于临床研究和药物开发研究。