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 66 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), 5-methylcytosine (m5C), N6-methyladenosine (m6A) nucleosides and mRNA containing Adenine- to-Inosine (A-to-I) modification at the site A of mouse 2C 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 2C receptor mRNA as well as A-to-I modifications found in other mRNA such as GluR2 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.