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 含有多种转录后核苷修饰，这些修饰在调节 RNA 功能中发挥着重要作用，并与多种人类疾病有关。 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 修饰图谱。不幸的是，由于RNA在体内的不稳定性及其非免疫原性，很难在动物体内产生针对RNA修饰的抗体。该项目的目标是开发一组单域抗体（sdAbs，也称为纳米抗体），能够利用 VHH 抗体噬菌体展示技术在单核苷酸水平上特异性识别 RNA 中的修饰核糖核苷。所提出的单域抗体具有非常高的亲和力、特异性、易于复制、稳定且尺寸非常小的优点，当与染料结合时能够实现超分辨率成像。在产生核苷的 sdAb 后，我们将通过 sdAb 与荧光染料的位点特异性偶联来开发荧光 sdAb。然后，我们将开发免疫印迹方法和方案，用于检测从多种生物/细胞中分离的 RNA 中的修饰核苷，并演示荧光 sdAb 在活细胞中修饰 RNA 体外成像中的应用。