Novel approach for rapid generation of human antibodies to RNA-modifications

快速生成针对 RNA 修饰的人类抗体的新方法

• 批准号: 9182930
• 负责人: ROLF K SWOBODA
• 金额: $ 22.46万
• 依托单位: ABZYME THERAPEUTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9182930
• 关键词: Adenine; Animals; Antibodies; Antibody Formation; Cell physiology; Cells; Clinical; DNA; Detection; Development; Electrophoretic Mobility Shift Assay; Engineering; Escherichia coli; Eukaryotic Cell; Generations; Genes; Goals; Human; Immunization; Immunoassay; Immunoprecipitation; In Vitro; Individual; Inosine; Investigation; Libraries; Messenger RNA; Modification; Monitor; Mus; Neuraxis; Nucleosides; Oligonucleotides; Oryctolagus cuniculus; Pathway interactions; Peptides; Performance; Phase; Phospho-Specific Antibodies; Phosphorylation; Physiology; Post-Transcriptional RNA Processing; Production; Protein Isoforms; Proteins; Proto-Oncogene Proteins c-akt; RNA; RNA Editing; RNA Instability; RNA Sequences; Reagent; Reporting; Research Personnel; Sampling; Schedule; Serotonin Receptor 5-HT2C; Signal Pathway; Signal Transduction; Site; Sorting - Cell Movement; Specificity; Structure; Surface; System; TimeLine; Tissues; Yeasts; base; expression cloning; immunogenicity; in vivo; interest; novel; novel strategies; protein activation; protein function; receptor; tumor

Project Summary: It is known that protein activity can be modulated by post-translational protein phosphorylation. Phospho-specific antibodies have greatly advanced understanding of the mechanisms of protein activation and signaling in cellular pathways. Post-transcriptional RNA modification is more complicated as there are 66 known RNA modifications that occur in thousands of RNAs in eukaryotic cells. A number of these RNA modifications have been shown to influence the development and physiology of the central nervous system (CNS). The availability of antibodies against RNA modifications at specific RNA sequences analogous to phospho-specific antibodies will facilitate investigation of the relationships between specific RNA modifications and their cellular function. Unfortunately due to in vivo instability of RNAs and their non-immunogenicity, it is difficult to raise antibodies against RNA modifications in animals. The goal of this project is to develop an in vitro novel platform for rapid generation and robust production of a set of antibodies against specific RNA modifications. As proof-of-principle, in Phase I, a yeast-based self-diversifying library of human single-chain variable fragment (ScFv) will be established and ScFv antibodies capable of recognizing Inosine (I), 5-methylcytosine (m5C), N6-methyladenosine (m6A) nucleosides and mRNA containing Adenine- to-Inosine (A-to-I) modification at the site A of mouse 2C receptor will be developed. In phase II, the effort will be dedicated to develop antibodies capable of recognizing RNA modification at a given RNA context such as A-to-I modification of A, B, C, D and E sites of mouse 2C receptor mRNA as well as A-to-I modifications found in other mRNA such as GluR2 and KA receptors. In addition, the developed platform will be used for generating antibodies to other RNA modifications. Availability of such reagents will enable researchers to monitor the effect of specific RNA modification on the structure or function of respective individual RNA or to analyze tissue sections, e.g. tumor samples, for the occurrence of such modifications.

项目概述：已知蛋白质的活性可以被翻译后蛋白质调控 磷酸化磷酸化特异性抗体极大地促进了对磷酸化的机制的理解。 蛋白质活化和细胞通路中的信号传导。转录后RNA修饰 在真核细胞中，有66种已知的RNA修饰发生在数千种RNA中。一 许多这些RNA修饰已被证明会影响发育和生理学， 中枢神经系统（CNS）。针对特定RNA修饰的抗体的可用性 类似于磷酸特异性抗体的序列将有助于研究 特定的RNA修饰及其细胞功能。不幸的是，由于RNA及其受体在体内的不稳定性， 由于RNA修饰是非免疫原性的，因此难以在动物中产生针对RNA修饰的抗体。这个目标 项目是开发一种体外新平台，用于快速生成和稳健生产一组抗体 针对特定的RNA修饰。作为原理的证明，在第一阶段，基于酵母的自我多样化文库， 将建立人单链可变片段（ScFv），并建立能够识别 肌苷（I）、5-甲基胞嘧啶（m5 C）、N6-甲基腺苷（m6 A）核苷和含有腺嘌呤的mRNA。 将开发小鼠2C受体位点A处的to-肌苷（A-to-I）修饰。在第二阶段， 致力于开发能够在给定RNA背景下识别RNA修饰的抗体， 小鼠2C受体mRNA的A、B、C、D和E位点的A至I修饰以及发现的A至I修饰 在其他mRNA中，如GluR 2和KA受体。此外，开发的平台将用于 产生针对其他RNA修饰的抗体。这些试剂的可用性将使研究人员能够 监测特异性RNA修饰对相应单个RNA的结构或功能的影响，或者 分析组织切片，例如肿瘤样品，以确定是否发生这种修饰。