Novel approach for rapid generation of human antibodies to RNA-modifications

Project Summary It is known that protein activity can be modulated by post translational protein phosphorylation Phospho specific antibodies have greatly advanced understanding of the mechanisms of protein activation and signaling in cellular pathways Post transcriptional RNA modification is more complicated as there are known RNA modifications that occur in thousands of RNAs in eukaryotic cells A number of these RNA modifications have been shown to influence the development and physiology of the central nervous system CNS The availability of antibodies against RNA modifications at specific RNA sequences analogous to phospho specific antibodies will facilitate investigation of the relationships between specific RNA modifications and their cellular function 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 an in vitro novel platform for rapid generation and robust production of a set of antibodies against specific RNA modifications As proof of principle in Phase I a yeast based self diversifying library of human single chain variable fragment ScFv will be established and ScFv antibodies capable of recognizing Inosine I methylcytosine m C N methyladenosine m A nucleosides and mRNA containing Adenine to Inosine A to I modification at the site A of mouse C receptor will be developed In phase II the effort will be dedicated to develop antibodies capable of recognizing RNA modification at a given RNA context such as A to I modification of A B C D and E sites of mouse C receptor mRNA as well as A to I modifications found in other mRNA such as GluR and KA receptors In addition the developed platform will be used for generating antibodies to other RNA modifications Availability of such reagents will enable researchers to monitor the effect of specific RNA modification on the structure or function of respective individual RNA or to analyze tissue sections e g tumor samples for the occurrence of such modifications Narrative As a result of this proposed project a novel antibody discovery platform will be established and a set of antibodies capable of detecting various RNA modifications will be developed These antibodies enable detection quantitation or immunoprecipitation of modified RNAs