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

• 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
• 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

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）：由阿达尔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。监测和操纵阿达尔酶和A-至-I RNA修饰的工具的可用性将促进特定A-至-I转化与其在CNS发育中的功能之间的关系的研究。该项目的目标是开发一套针对A-to-I RNA编辑酶及其修饰RNA产物的纳米抗体。预期这些纳米抗体不仅用作检测、定量或免疫沉淀的试剂，而且用作细胞内抗体 能够抑制靶阿达尔酶的活性或与细胞中特异性A至I修饰的RNA结合。作为原理证明，在第I阶段，使用Abzyme专有的自我多样化单结构域抗体文库平台进行高通量抗体发现，将开发针对含肌苷的通用RNA和小鼠血清素2C受体的位点特异性A-to-I mRNA同种型的纳米抗体，以及ADAR 2中和纳米抗体。在II期，将产生针对小鼠ADAR 1和ADAR 3以及其他CNS突出的A至I修饰RNA的纳米抗体。此外，将用CNS特异性和按需表达的ADAR 2胞内抗体产生转基因敲入小鼠，并研究ADAR 2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阿达尔酶或特异性修饰的RNA的功能。