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

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

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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.

项目摘要/摘要 RNA的转录后修饰对于RNA稳定性，运输和翻译至关重要蛋白质。在人类中，最普遍的RNA修饰的破坏是N6-甲基腺苷（M6A）与癌症等疾病有关。为了更好地了解M6A在疾病发病机理中的作用，生物学家需要强大的方法来映射RNA中的M6A位点。尽管最近进展，现有方法需要许多步骤，缺乏选择性和灵敏度，或表现出序列偏差。该项目的长期目标是通过开发可以检测M6A并将这些工具应用这些工具的酶来解决这些限制癌细胞的表面参考分析。将同时采用两种策略：1）进化反向 - 转录酶，使用RNA作为模板聚合DNA，将核苷酸“标记”掺入DNA中在细胞RNA的逆转录过程中，在M6A位点。这将有助于直接读数M6A 基于抗体的下拉的要求。 2）工程师脱氨酶标记M6A位点。腺嘌呤脱氨酶将腺嘌呤（a）转换为插入（i），该（i）被聚合酶读为鸟嘌呤（g）。那是工程脱氨酶为了有选择地脱氨酸M6A而不是A，将导致可以通过RNA测序定位的标记。最后，进化的逆转录酶和脱氨酶平台将在生物化学上彻底进行为哺乳动物细胞的M6A测序进行表征和实施，以建立所提出的平台。这这些工具的开发和传播将通过促进对M6A的研究及其作用产生广泛的影响在人类健康方面。