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

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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.