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

RNA含有多种转录后核苷修饰，这些修饰在调节RNA功能中起重要作用，并且与各种人类疾病有关。 为了破译RNA修饰的功能，非常需要强大的分析工具，使科学家能够检测，监测和绘制RNA修饰。不幸的是，由于RNA的体内不稳定性和它们的非免疫原性，很难在动物中产生针对RNA修饰的抗体。该项目的目标是利用VHH抗体噬菌体展示技术开发一组能够在单核苷酸水平特异性识别RNA中修饰的核糖核苷的单域抗体（sdAb，也称为纳米抗体）。所提出的单结构域抗体具有非常高的亲和力、特异性的优点，并且它们易于复制、稳定且尺寸非常小，当与染料缀合时能够实现超分辨率成像。在产生针对核苷的sdAb之后，我们将通过将sdAb与荧光染料位点特异性偶联来开发荧光sdAb。然后，我们将开发用于检测从各种生物体/细胞分离的RNA中修饰的核苷的免疫印迹方法和方案，并展示荧光sdAb用于活细胞中修饰的RNA的体外成像的应用。

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.