Nanobodies for detecting and manipulating A to I editing enzymes and their modified RNA products

用于检测和操纵A至I编辑酶及其修饰的RNA产物的纳米抗体

基本信息

批准号：
8841496
负责人：
Hiep T Tran
金额：
$ 22.5万
依托单位：
ABZYME THERAPEUTICS, LLC
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8841496
关键词：
ADAR1 Adenosine Amino Acid Sequence Animals Anorexia Nervosa Antibodies Antigens Anxiety Binding Biological Process Catalytic Antibodies Cell Line Cell Separation Cells Codon Nucleotides Custom DRADA2b protein Desire for food Detection Development Enzyme-Linked Immunosorbent Assay Enzymes Escherichia coli Etiology Family Fluorescence Future Generations Generic Drugs Genes Genetic Genome Goals Immunoassay Immunoprecipitation In Vitro Individual Inosine Inosine Nucleotides Investigation Ion Channel Knock-in Mouse Laboratories Libraries Locomotion Mediating Mental Depression Mental disorders Messenger RNA Modification Monitor Moods Mus Mutation Neuraxis Obsessive-Compulsive Disorder Performance Phase Process Production Property Protein Family Protein Isoforms Proteins RNA RNA Editing RNA-specific adenosine deaminase 3 Reagent Recombinant Proteins Reporter Reporting Research Personnel Role Schedule Serotonin Serotonin Receptor 5-HT2C Services Sex Behavior Site Sorting - Cell Movement Specificity Surface System Task Performances Terminator Codon Time TimeLine Transgenic Animals Transgenic Organisms Tryptophan Validation Western Blotting Yeasts adenosine deaminase assault base expression cloning nanobodies nervous system development neurophysiology public health relevance receptor tool

项目摘要

DESCRIPTION (provided by applicant): Adenosine to inosine or A-to-I RNA editing executed by ADAR family proteins is abundant in the central nervous system (CNS). The availability of tools to monitor and manipulate both ADAR enzymes and A-to-I RNA modifications will facilitate investigation of the relationships between specific A-to-I conversions and their function in CNS development. The goal of this project is to develop a set of nanobodies to A-to-I RNA editing enzymes and their modified RNA products. These nanobodies are expected to serve not only as reagents for detection, quantification, or immunoprecipitation but also as intracellular antibodies capable of inhibiting the activity of target ADAR enzymes or binding to specific A-to-I modified RNAs in cells. As proof-of-principle, in Phase I, using Abzyme's proprietary self-diversifying single domain antibody library platform for high throughput antibody discovery, nanobodies to inosine-containing generic RNAs and site- specific A-to-I mRNA isoforms of mouse serotonin 2C receptor, as well as ADAR2-neutralizing nanobodies will be developed. In Phase II, nanobodies to mouse ADAR1 and ADAR3 as well as other CNS prominent A-to- I modified RNAs will be generated. In addition, transgenic knock-in mice will be generated with CNS-specific and expression-on-demand intrabodies to ADAR2, and the temporal impact of ADAR2 inactivation on CNS development will be investigated. The availability of such nanobodies will facilitate development of both immunoassays and cell lines or transgenic animals in which the function of ADAR enzymes or specific modified RNA can be investigated in time and space.

描述（由申请人提供）： ADAR 家族蛋白执行的腺苷至肌苷或 A 至 I RNA 编辑在中枢神经系统 (CNS) 中丰富。监测和操作 ADAR 酶和 A 至 I RNA 修饰的工具的可用性将有助于研究特定的 A 至 I 转换及其在中枢神经系统发育中的功能之间的关系。该项目的目标是开发一套针对A-to-I RNA编辑酶的纳米抗体及其修饰的RNA产物。这些纳米抗体不仅可用作检测、定量或免疫沉淀的试剂，还可用作细胞内抗体能够抑制目标 ADAR 酶的活性或与细胞中特定的 A-to-I 修饰 RNA 结合。作为原理验证，在第一阶段，使用 Abzyme 专有的自我多样化单域抗体库平台进行高通量抗体发现、含有肌苷的通用 RNA 和小鼠血清素 2C 的位点特异性 A-to-I mRNA 亚型的纳米抗体受体以及 ADAR2 中和纳米抗体将被开发。在第二阶段，将产生针对小鼠 ADAR1 和 ADAR3 以及其他 CNS 突出的 A 至 I 修饰 RNA 的纳米抗体。此外，将产生具有 CNS 特异性和按需表达 ADAR2 体内抗体的转基因敲入小鼠，并将研究 ADAR2 失活对 CNS 发育的时间影响。这种纳米抗体的可用性将促进免疫测定和细胞系或转基因动物的开发，其中可以在时间和空间上研究 ADAR 酶或特定修饰 RNA 的功能。