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

RNA含有各种各样的转录后核苷修饰，它们在调节RNA功能方面发挥着重要作用，并与各种人类疾病有关。为了破译RNA修饰的功能，迫切需要强大的分析工具来使科学家能够检测、监测和绘制RNA修饰图。遗憾的是，由于RNA在体内的不稳定性和非免疫原性，在动物体内很难产生针对RNA修饰的抗体。本项目的目标是利用VHH抗体噬菌体展示技术，开发一套能够在单核苷酸水平上特异性识别RNA中修饰的核糖核苷的单域抗体(sdAbs，又称纳米体)。所提出的单域抗体具有非常高的亲和力和特异性，并且它们易于复制，稳定，并且尺寸非常小，当连接到染料时能够进行超分辨率成像。在制备针对核苷的sdAbbs后，我们将通过将sdAbbs与荧光染料进行定点偶联来开发荧光sdAbbs。然后，我们将开发免疫印迹方法和方案来检测从各种生物/细胞中分离的RNA中的修饰核苷，并展示荧光sdAbs在体外对活细胞中的修饰RNA进行成像的应用。

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.