Multi-epitope tag (MET)-recombinant antibody toolbox for detection and manipulation of RNA modifications

Summary RNA plays a central role in numerous cellular processes Over RNA modifications have been characterized of which twelve have been identified in messenger RNA mRNA With the exception of the mRNA cap structure little is known about their function The discovery that the FTO gene initially linked to obesity and energy homeostasis is an oxidative demethylase eraser of methyl adeonsine m A in RNA has spurred interest in understanding the biological function of RNA modifications and how their dysregulation may impact disease The identification of hydroxymethyl adenine hm A and formyl adensosine f A as demethylation intermediates with the potential for independent biological function has resulted in the coining of the terms epitranscriptome and epi modifications drawing comparison to the dynamic regulation of methylcytosine m C and hydroxymethylcytosine hm C in DNA The epitranscriptome field is still in its discovery phase with an unmet need for highly sensitive and specific reagents for the visualization isolation and analysis of the biological function of RNA epi modifications This Phase I proposal intends to develop and validate an RNA epi modification multi epitope tag MET recombinant antibody toolbox wherein the antibody heavy chain is engineered to contain the sequences recognized by the protein ligase SortaseA and the biotin ligase BirA thereby enabling targeted end user customizable labeling XHis tag is also present enabling purification of the functionalized antibody under mild conditions SortaseA can be used to attach a large repertoire of payloads ranging from fluorescent dyes to bioactive peptides Targeted biotinylation enables leveraging of avidin streptavidin based technologies for maximizing detection sensitivity or isolation efficiency in enrichment based detection methods Aim efforts will develop a specificity validation pipeline using existing RNA modification antibodies and DNA modification antibodies whose targets are also found in RNA In Aim recombinant MET antibodies will be generated to two RNA epi modifications using the Aim specificity pipeline to identify highly specific antibodies Aim efforts will establish proof of concept for the targeted conjugation of MET antibodies to a fluorophore using Sortase A or to biotin using BirA Future Phase II efforts will refine these methodologies and will be expand to include development of kits and reagents for end user customized antibody conjugates and assay systems for the visualization isolation and analysis of RNA epi modifications in a variety of cell and tissue types These antibodies and kits will serve as enabling tools allowing the discovery of the fundamental mechanisms that regulate RNA epigenetics and could eventually be used in clinical or diagnostic applications Project Narrative While the biological significance of chemical modifications found on RNA molecules and their interacting proteins is still in its discovery phase already it is known that the dysfunction of these proteins are involved in obesity mental retardation and possibly substance abuse This grant proposes to develop a highly specific and sensitive tool kit of antibodies that will allow for the visualization isolation and study of these biomolecules which will further our understanding of their role in human diseases