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）：中枢神经系统（CNS）中丰富了肌苷腺苷或A-To-I RNA编辑。监测和操纵ADAR酶和A-to-I RNA修改的工具的可用性将有助于研究特定的A-TO-I转换及其在中枢神经系统开发中的功能之间的关系。该项目的目的是为A-to-I RNA编辑酶及其修饰的RNA产品开发一组纳米体。这些纳米型不仅可以用作检测，定量或免疫沉淀的试剂，而且还作为细胞内抗体能够抑制靶向ADAR酶的活性或与细胞中特定A-至I修饰的RNA结合。作为原则证明，在第一阶段，使用Abzyme的专有自变化的单个结构域抗体库平台用于高吞吐量抗体发现，对含肌苷的通用RNA的纳米构造以及小鼠羟色胺2C受体的纳米构造，特定的A-TO-I mRNA同工型小鼠2C受体和adar2-natrabsies Nan nanobodies nan nanobodies Nanabodies nan nanobodies Nan nanobodies Nanobodies。在II阶段，将生成对小鼠ADAR1和ADAR3的纳米体系以及其他CNS突出的A到I修改的RNA。此外，将使用CNS特异性和对ADAR2表达表达的内形式表达的转基因敲击小鼠产生，并且将研究ADAR2失活对CNS发育的时间影响。这种纳米生物的可用性将促进免疫测定和细胞系或转基因动物的发展，其中可以在时间和空间中研究ADAR酶或特定修饰的RNA的功能。