Multi-epitope tag (MET)-recombinant antibody toolbox for detection and manipulation of RNA modifications

用于检测和操作 RNA 修饰的多表位标签 (MET) 重组抗体工具箱

• 批准号: 9183142
• 负责人: Laura Rocco Carpenter
• 金额: $ 22.5万
• 依托单位: ACTIVE MOTIF, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9183142
• 关键词: Address; Adenine; Adenosine; Amino Acid Sequence; Antibodies; Antigens; Avidin; Biological; Biological Assay; Biological Process; Biotin; Biotinylation; Brain; Cell physiology; Chemicals; Chemistry; Clinical; Coin; DNA; DNA Methylation; DNA Modification Process; DNA Sequence; Detection; Development; Diagnostic; Discrimination; Disease; Engineering; Enzyme-Linked Immunosorbent Assay; Enzymes; Epigenetic Process; Epitopes; Evaluation; Feasibility Studies; Fertility; Fluorescent Dyes; Functional disorder; Future; Gene Duplication; Genes; Goals; Grant; Histocompatibility Testing; Homeostasis; Human; Hybridomas; Imagery; Immunofluorescence Immunologic; Immunoglobulin Variable Region; Impairment; In Vitro; Knowledge; Label; Ligase; Light; Link; Mental Retardation; Messenger RNA; Metabolism; Methodology; Methods; Microcephaly; Midbrain structure; Modification; Monoclonal Antibodies; Mus; Neurons; Obesity; Peptide Sequence Determination; Peptides; Phase; Pilot Projects; Play; Post-Translational Protein Processing; Proteins; Protocols documentation; RNA; RNA Caps; RNA analysis; RNA immunoprecipitation sequencing; Reader; Reagent; Recombinant Antibody; Recombinants; Regulation; Research; Ribonucleotides; Role; Site; Site-Directed Mutagenesis; Specificity; Streptavidin; Structure; Substance abuse problem; System; Technology; Testing; Time; Transcript; Transfection; Validation; aerobic respiration control protein; antibody conjugate; base; cell type; cost; demethylation; dopaminergic neuron; expression cloning; fluorophore; human disease; in vivo; interest; loss of function mutation; novel; polyclonal antibody; screening; sortase; tissue/cell culture; tool

Summary RNA plays a central role in numerous cellular processes. Over 100 RNA modifications have been characterized, of which twelve have been identified in messenger RNA (mRNA). With the exception of the mRNA cap structure, little is known about their function. The discovery that the FTO gene, initially linked to obesity and energy homeostasis, is an oxidative demethylase (“eraser”) of methyl-6-adeonsine (m6A) in RNA has spurred interest in understanding the biological function of RNA modifications and how their dysregulation may impact disease. The identification of hydroxymethyl-6-adenine (hm6A) and formyl-6-adensosine (f6A) as demethylation intermediates with the potential for independent biological function has resulted in the coining of the terms “epitranscriptome” and “epi-modifications”, drawing comparison to the dynamic regulation of 5- methylcytosine (m5C) and 5-hydroxymethylcytosine (hm5C) in DNA. The epitranscriptome field is still in its discovery phase, with an unmet need for highly sensitive and specific reagents for the visualization, isolation and analysis of the biological function of RNA epi-modifications. This Phase I proposal intends to develop and validate an RNA epi-modification multi-epitope tag (MET) recombinant antibody “toolbox” wherein the antibody heavy chain is engineered to contain the sequences recognized by the protein ligase SortaseA and the biotin ligase BirA, thereby enabling targeted, end-user customizable labeling. 6XHis tag is also present, enabling purification of the functionalized antibody under mild conditions. SortaseA can be used to attach a large repertoire of payloads ranging from fluorescent dyes to bioactive peptides. Targeted biotinylation enables leveraging of avidin/streptavidin-based technologies for maximizing detection sensitivity or isolation efficiency in enrichment-based detection methods. Aim 1 efforts will develop a specificity validation pipeline using existing RNA modification antibodies and DNA modification antibodies whose targets are also found in RNA. In Aim 2, recombinant MET antibodies will be generated to two RNA epi-modifications using the Aim 1 specificity pipeline to identify highly specific antibodies. Aim 3 efforts will establish proof of concept for the targeted conjugation of MET antibodies to a fluorophore using Sortase A or to biotin using BirA. Future Phase II efforts will refine these methodologies and will be expand to include development of kits and reagents for end-user customized antibody conjugates and assay systems for the visualization, isolation and analysis of RNA epi- modifications in a variety of cell and tissue types. These antibodies and kits will serve as enabling tools allowing the discovery of the fundamental mechanisms that regulate RNA epigenetics and could eventually be used in clinical or diagnostic applications.

摘要 RNA在许多细胞过程中起着核心作用。已经有100多种RNA修饰被发现 其中12个已在信使RNA(MRNA)中被鉴定。除了 关于其结构，人们对其功能知之甚少。发现FTO基因最初与 肥胖与能量平衡，是RNA中甲基-6-脱氧核糖核酸(M6A)的一种氧化去甲基酶 激发了人们对了解RNA修饰的生物学功能以及它们的失调是如何 可能会影响疾病。羟甲基-6-腺嘌呤(Hm6A)和甲酰-6-腺苷(F6A)的鉴定 具有独立生物功能的去甲基化中间体导致了 术语“表位转录组”和“表位修饰”，将其与5- DNA中的甲基胞嘧啶(M5C)和5-羟甲基胞嘧啶(Hm5C)。Epecranscritome字段仍在其 发现阶段，对用于可视化、分离的高度敏感和特定试剂的需求未得到满足 并对RNA表观修饰的生物学功能进行了分析。此第一阶段提案旨在开发和 验证RNA表修饰多表位标签(MET)重组抗体“工具箱”，其中该抗体 重链被设计成包含由蛋白质连接酶SortaseA和生物素识别的序列 连接Bira，从而实现有针对性的、终端用户可定制的标签。6XHis标签也存在，启用 温和条件下功能化抗体的纯化。SortaseA可用于将大型 有效载荷范围从荧光染料到生物活性多肽。靶向生物素化使 利用基于亲和素/链霉亲和素的技术最大限度地提高检测灵敏度或分离效率 在基于浓缩的检测方法中。AIM 1的努力将开发一条特异性验证管道，使用 现有的RNA修饰抗体和DNA修饰抗体，其靶标也在RNA中发现。在……里面 目的2，利用目的1的特异性产生针对两个rna表位修饰的重组MET抗体。 鉴定高度特异性抗体的管道。目标3的努力将为目标建立概念证明 使用山梨酸酶A与荧光团的MET抗体或使用BIRA与生物素的偶联。未来第二阶段的工作 将完善这些方法，并将扩大到包括为最终用户开发试剂盒和试剂 定制的抗体结合物和检测系统用于显示、分离和分析RNAepi- 在各种细胞和组织类型中的修饰。这些抗体和试剂盒将成为使能工具 允许发现调节RNA表观遗传学的基本机制，并最终可能 用于临床或诊断应用。