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

总结 RNA在许多细胞过程中起着核心作用。已经有超过100种RNA修饰， 其特征在于，其中12个已在信使RNA（mRNA）中被鉴定。两岸除了 mRNA帽结构，对其功能知之甚少。发现FTO基因，最初与 肥胖和能量稳态，是RNA中甲基-6-腺苷（m6 A）的氧化脱甲基酶（“橡皮擦”） 这激发了人们对理解RNA修饰的生物学功能以及它们的失调是如何发生的兴趣。 可能影响疾病。羟甲基-6-腺嘌呤（hm 6A）和甲酰基-6-腺苷（f6 A）的结构鉴定 具有独立生物学功能潜力的去甲基化中间体导致了 术语“epitranscriptome”和“epi-modifications”，与5- 甲基胞嘧啶（m5 C）和5-羟甲基胞嘧啶（hm 5C）。epitranscriptome领域仍处于其 发现阶段，需要高度敏感和特异性的试剂，用于可视化、分离 以及RNA表观修饰的生物学功能分析。第一阶段计划旨在开发和 验证RNA表位修饰多表位标签（MET）重组抗体“工具箱”， 重链被工程化以含有由蛋白质连接酶分选酶A和生物素识别的序列 连接酶BirA，从而实现有针对性的，最终用户可定制的标记。6XHis标签也存在， 在温和条件下纯化功能化抗体。SortaseA可用于连接大型 从荧光染料到生物活性肽。靶向生物素化使 利用基于抗生物素蛋白/链霉抗生物素蛋白的技术来最大化检测灵敏度或分离效率 在基于富集的检测方法中。目标1的努力将开发一个特异性验证管道， 现有的RNA修饰抗体和DNA修饰抗体，其靶也存在于RNA中。在 目的2，利用Aim 1特异性，将重组MET抗体产生为两个RNA表位修饰 用于识别高度特异性抗体的管道。目标3的努力将为目标建立概念验证， 使用分选酶A将MET抗体缀合至荧光团或使用BirA缀合至生物素。未来第二阶段的工作 将改进这些方法，并将扩大到包括为最终用户开发成套工具和试剂 定制的抗体偶联物和测定系统，用于RNA表位的可视化、分离和分析， 在多种细胞和组织类型中的修饰。这些抗体和试剂盒将作为使能工具 允许发现调节RNA表观遗传学的基本机制，并最终可能成为 用于临床或诊断应用。

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.