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

• 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
• 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; 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

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.

项目摘要 急性转录反应是从发育到 学习和记忆，并被认为是药物使用障碍背后的推动力。 但是，当前测量转录急性变化的测定缺乏对功率的敏感性和吞吐量 对神经元刺激的直接转录反应的详细机械研究。广泛使用的mRNA- SEQ分析通过量化稳定的聚腺苷酸化RNA的大体水平来测量转录，该RNA早期掩盖 转录响应。 mRNA-SEQ也无法捕获非蛋白质编码RNA，例如增强剂RNA （ERNAS），与增强子活性相关，并在神经元中增强子功能中起重要作用。 测量新合成的“新生RNA”的测定能够测量转录的早期变化， 包括Ernas。但是，当前测量新生RNA合成的测定需要复杂的工作流程，并且 表现出较差的评估敏感性，从而限制了它们用于神经科学研究的使用。而不是测量新生的RNA，而是 RNA聚合酶的直接测量提供了一种强大的研究转录方法 这些限制。 RNA聚合酶II（RNAPII）是转录蛋白质编码mRNA和的聚合酶 非编码RNA，包括ERNAS，并包含一个偶有的羧基末端结构域（CTD） 被磷酸化以促进转录启动和伸长。映射RNAPII和 它的磷酸剂为直接测量转录动态提供了独特的机会，但是当前的测定法 绘制rnapii（例如chip-seq）缺乏实现此目的的敏感性。 在这里，Epicypher正在开发Cutana-Rnapii，这是一种直接的RNAPII映射测定法 量化全球转录动力学，以响应神经科学研究和药物的细胞外刺激 发展。我们提案的创新是开发针对的修改后的剪切和标签工作流程 各种RNAPII CTD磷酸化位点，以测量转录启动和伸长的早期变化。 该阶段I可行性研究的目的是证明Cutana-RNAPII测定的灵敏度以测量 神经元中的早期转录反应。在AIM 1中，我们将开发Cutana-rnapii工作流程和 基准我们对当前金色标准的新生RNA分析（Pro-Seq分析）进行评估以测量 响应赛酮治疗的细胞中的转录动力学。在AIM 2中，我们将与Zoe McElligott博士合作 为了证明Cutana-Rnapii测量神经科学早期转录动力学的能力 阿片类药物暴露的模型。在第一阶段成功之后，在第二阶段，我们将发展强大的cutana-rnapii β套件和自动化测定服务，使用低样本输入（包括） 单细胞。这项工作将是部署cutana-rnapii来研究高度异构脑样本的关键 用于突破性的神经科学和药物使用研究。这个低成本的测定平台将彻底改变 可以在复杂的组织中研究转录动力学，以进行临床研究和药物开发研究。