RNA plays a central role in numerous cellular processes. Over 100 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-6-adeonsine (m6A) in RNA has spurred interest in understanding the biological function of RNA modifications and how their dysregulation may impact disease. The identification of hydroxymethyl-6-adenine (hm6A) and formyl-6-adensosine (f6A) 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 5- methylcytosine (m5C) and 5-hydroxymethylcytosine (hm5C) 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. 6XHis 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 1 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 2, recombinant MET antibodies will be generated to two RNA epi-modifications using the Aim 1 specificity pipeline to identify highly specific antibodies. Aim 3 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.
Public Health Relevance Statement: 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.
Project Terms: Address; Adenine; Adenosine; aerobic respiration control protein; Amino Acid Sequence; Antibodies; antibody conjugate; Antigens; Avidin; base; Biological; Biological Assay; Biological Process; Biotin; Biotinylation; Brain; Cell physiology; cell type; Chemicals; Chemistry; Clinical; Coin; cost; demethylation; Detection; Development; Diagnostic; Discrimination; Disease; DNA; DNA Methylation; DNA Modification Process; DNA Sequence; dopaminergic neuron; Engineering; Enzyme-Linked Immunosorbent Assay; Enzymes; Epigenetic Process; Epitopes; Evaluation; expression cloning; Feasibility Studies; Fertility; Fluorescent Dyes; fluorophore; Functional disorder; Future; Gene Duplication; Genes; Goals; Grant; Histocompatibility Testing; Homeostasis; Human; human disease; Hybridomas; Imagery; Immunofluorescence Immunologic; Immunoglobulin Variable Region; Impairment; In Vitro; in vivo; interest; Knowledge; Label; Ligase; Light; Link; loss of function mutation; Mental Retardation; Messenger RNA; Metabolism; Methodology; Methods; Microcephaly; Midbrain structure; Modification; Monoclonal Antibodies; Mus; Neurons; novel; Obesity; Peptide Sequence Determination; Peptides; Phase; Pilot Projects; Play; polyclonal antibody; Post-Translational Protein Processing; Proteins; Protocols documentation; Reader; Reagent; Recombinant Antibody; Recombinants; Regulation; Research; Ribonucleotides; RNA; RNA analysis; RNA Caps; RNA immunoprecipitation sequencing; Role; screening; Site; Site-Directed Mutagenesis; sortase; Specificity; Streptavidin; Structure; Substance abuse problem; System; Technology; Testing; Time; tissue/cell culture; tool; Transcript; Transfection; Validation
