Directed Evolution of an Epitranscriptomic Toolkit

表观转录组工具包的定向进化

基本信息

批准号：
10703449
负责人：
Monica Neugebauer
金额：
$ 7.18万
依托单位：
BROAD INSTITUTE, INC.
依托单位国家：
美国
项目类别：
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/10703449
关键词：
Address Adenine Adenosine Antibodies Area Base Pairing Binding Sites 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 Maps 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 Reverse engineering Role Site Site-Directed Mutagenesis Techniques Testing Thymidine Training Translations Tube adenosine deaminase cancer cell career cost detection method epitranscriptomics genetic information human disease interest operation skills success therapeutic target tool trafficking transcriptome sequencing

项目摘要

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在人类中，最常见的 RNA 修饰 N6-甲基腺苷 (m6A) 的破坏已被证实。为了更好地了解 m6A 在疾病发病机制中的作用，生物学家进行了研究。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，但现有方法仍然需要步骤众多，缺乏选择性和敏感性，或表现出序列偏差该项目的长期目标。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同时进行：1）逆向进化。转录酶，使用 RNA 作为模板聚合 DNA，将核苷酸“标记”整合到 DNA 中在细胞 RNA 逆转录过程中位于 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。将腺嘌呤 (A) 转化为肌苷 (I)，聚合酶将其解读为鸟嘌呤 (G)，从而设计脱氨酶。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。