IGF::OT::IGF Single domain antibodies for detection and imaging of modified RNA nucleosides

IGF::OT::IGF 单域抗体，用于修饰 RNA 核苷的检测和成像

• 批准号: 9571729
• 负责人: XICHUN ZHOU
• 金额: $ 24.99万
• 依托单位: VITAN-BIOTECH, LLC
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-18 至 2018-06-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9571729
• 关键词: Affinity; Animals; Antibodies; Binding; Biotin; C-terminal; Cells; Coupling; Cysteine; Data Set; Detection; Development; Dyes; Engineering; Epigenetic Process; Epitopes; Fluorescent Dyes; Genetic Transcription; Goals; Haptens; Image; Immune; Immunoblotting; In Vitro; Label; Link; Maps; Methods; Modification; Monitor; Nucleosides; Nucleotides; Organism; Phage Display; Phase; Play; Protocols documentation; Pseudouridine; RNA; RNA Instability; Research; Resolution; Ribonucleosides; Role; Scientist; Site; Specificity; Technology; analytical tool; cyanine dye 5; human disease; in vivo; nanobodies; nanomolar; tool

RNAs contain a wide variety of post-transcriptional nucleoside modifications which play important roles in modulating RNA functions and are linked to various human diseases. To decipher the functions of RNA modifications, robust analytical tools that enable scientists to detect, monitor, and map RNA modifications are highly needed. 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 a set of single domain antibodies (sdAbs, also called nanobodies) capable of specifically recognizing modified ribonucleosides in RNA at the single nucleotide level using VHH antibody phage display technology. The proposed single-domain antibodies have the advantages of very high affinity, specificity, and they are easy to reproduce, stable, and of very small size, enabling super-resolution imaging when conjugated to dye. After the sdAbs to nucleosides are produced, we will develop fluorescent sdAbs via a site-specific coupling the sdAbs with fluorescent dyes. We will then develop immuno-blotting method and protocols for the detection of modified nucleosides from RNAs isolated from a variety of organisms/cells, and demonstrate the applications of the fluorescent sdAbs for in vitro imaging the modified RNAs in living cells.

RNA含有各种各样的转录后核苷修饰，这些修饰在调节RNA功能中起重要作用，并与各种人类疾病有关。为了破译RNA修饰的功能，科学家们非常需要强大的分析工具来检测、监测和绘制RNA修饰。不幸的是，由于RNA在体内的不稳定性和它们的非免疫原性，很难在动物体内培养针对RNA修饰的抗体。该项目的目标是开发一套单域抗体（sabs，也称为纳米体），能够在单核苷酸水平上使用VHH抗体噬菌体展示技术特异性识别RNA中修饰的核糖核苷。所提出的单域抗体具有亲和力高、特异性强、易于复制、稳定性好、体积小等优点，结合染料后可实现超分辨率成像。在核酸单克隆抗体产生后，我们将通过与荧光染料的位点特异性偶联来开发荧光单克隆抗体。然后，我们将开发免疫印迹方法和方案，用于检测从各种生物体/细胞中分离的rna中修饰的核苷，并演示荧光sabs在活细胞中修饰rna的体外成像应用。