Directed Evolution of an Epitranscriptomic Toolkit

表观转录组工具包的定向进化

• 批准号: 10312494
• 负责人: Monica Neugebauer
• 金额: $ 6.6万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10312494
• 关键词: Address; Adenine; Adenosine; Antibodies; Area; Base Pairing; Biochemical; Biological; Cell Culture Techniques; Cell Line; Cell physiology; Cells; Cellular biology; Chemicals; DNA; Deaminase; Deamination; Detection; Development; Directed Molecular Evolution; Disease; Engineering; Enzymes; Evolution; Generations; Genetic Transcription; Goals; Guanine; Health; Human; In Vitro; Inosine; Libraries; Link; Location; Malignant Neoplasms; Mammalian Cell; Messenger RNA; Methods; Methylation; Modification; Nucleotides; Pathogenesis; Play; Polymerase; Polymers; Post-Transcriptional RNA Processing; Probability; Proteins; RNA; RNA Stability; RNA analysis; RNA-Directed DNA Polymerase; Reagent; Regulation; Research; Research Personnel; Resolution; Reverse Transcription; Role; Site; Site-Directed Mutagenesis; Techniques; Testing; Thymidine; Training; Translations; Tube; adenosine deaminase; base; cancer cell; career; cost; detection method; epitranscriptomics; genetic information; human disease; interest; operation; skills; success; therapeutic target; tool; trafficking; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT The post-transcriptional modification of RNA is critical for RNA stability, trafficking, and translation into proteins. In humans, disruption of the most prevalent RNA modification, N6-methyladenosine (m6A), has been linked to diseases such as cancer. To better understand the role of m6A in disease pathogenesis, biologists require robust methods for mapping m6A sites within RNA. Despite recent progress, existing methods require numerous steps, lack selectivity and sensitivity, or demonstrate sequence bias. The long-term goal of this project is to address these limitations by developing enzymes that can detect m6A and applying these tools for epitranscriptomic analysis of cancer cells. Two strategies will be pursued in parallel: 1) Evolve reverse- transcriptases, which polymerize DNA using RNA as a template, to incorporate a nucleotide “marker” into DNA at m6A sites during reverse transcription of cellular RNA. This would facilitate direct readout of m6A without the requirement for antibody-based pulldowns. 2) Engineer deaminases to mark m6A sites. Adenine deaminases convert adenine (A) to inosine (I), which is read as guanine (G) by polymerases. Thus, engineering deaminases to selectively deaminate m6A, rather than A, would result in a mark that can be located by RNA sequencing. Finally, the evolved reverse transcriptase and deaminase platforms will be thoroughly biochemically characterized and implemented for m6A sequencing of mammalian cells to establish the proposed platform. The development and dissemination of these tools will have a broad impact by facilitating studies of m6A and its role in human health.

项目概要/摘要 RNA 的转录后修饰对于 RNA 的稳定性、运输和翻译至关重要 蛋白质。在人类中，最常见的 RNA 修饰 N6-甲基腺苷 (m6A) 的破坏已被证实 与癌症等疾病有关。为了更好地了解 m6A 在疾病发病机制中的作用，生物学家 需要稳健的方法来绘制 RNA 内的 m6A 位点。尽管最近取得了进展，但现有方法需要 步骤众多，缺乏选择性和敏感性，或表现出序列偏差。本项目的长期目标 的目的是通过开发可以检测 m6A 的酶并将这些工具应用于 癌细胞的表观转录组分析。将并行实施两种策略：1）逆向发展 转录酶，使用 RNA 作为模板聚合 DNA，将核苷酸“标记”整合到 DNA 中 细胞 RNA 逆转录期间的 m6A 位点。这将有助于直接读出 m6A，而无需 基于抗体的下拉的要求。 2) 设计脱氨酶来标记 m6A 位点。腺嘌呤脱氨酶 将腺嘌呤 (A) 转化为肌苷 (I)，聚合酶将肌苷 (I) 读取为鸟嘌呤 (G)。因此，工程脱氨 选择性地脱氨基 m6A（而不是 A）将产生可通过 RNA 测序定位的标记。 最后，进化的逆转录酶和脱氨酶平台将被彻底生化 对哺乳动物细胞的 m6A 测序进行了表征和实施，以建立拟议的平台。这 这些工具的开发和传播将促进 m6A 及其作用的研究，从而产生广泛的影响 在人类健康方面。