A Library of Immunoaffinity Reagents for RNA modifications

Project Abstract While there are commercial monoclonal antibodies against several RNA modifications there is a general paucity of immunological tools for the andgt known RNA modifications This lack of immunological tools is hindering study of the emerging roles for modified ribonucleosides as critical players in the many basic cell and organismal processes in viruses bacteria parasites and single cell eukaryotes as well as in human health and disease Furthermore the specificity of available antibodies has not been rigorously or systematically validated against most other RNA modifications To meet this need we propose to exploit our expertise in antibody development and in nucleic acid modification chemistry and biology to pilot the development of a library of scFv antibody fragments capable of specifically binding modified ribonucleosides in RNA at the single nucleotide level Recombinant antibodies will be identified using a novel yeast display platform building off of murine immunization with a panel of ribonucleosides These ribonucleosides were selected both because of their biological relevance and because they are of interest to the research community Furthermore we propose to fine tune specificity and affinity by engineering the individual recombinant antibodies to recognize related structures using our paratope epitope optimization technology The resulting recombinant antibodies will be validated for common end user applications such as immunoprecipitation ELISAs and Northwestern blots We anticipate that there will be numerous applications for high quality antibody reagents including affinity purification of modified RNA species immunohistochemistry in cells and tissue sections Northwestern blots of total RNA and diagnostic tools for RNA modification based disease models Development and availability of immunological reagents has revolutionized many areas of biochemistry including examining and quantifying signaling pathways protein localization within the cell and intracellular trafficking among others We propose to couple ribonucleoside chemistry with state of the art library construction and antibody engineering to develop specific high affinity antibody based reagents for modified ribonucleosides