Developing a Chemical Proteomics Platform for Capturing and Liganding RNA-binding Activity in Cells

Analysis of the proteome using mass spectrometry enables unparalleled information about cellular function. Small molecule covalent probes can survey protein activity through activity-based protein profiling (ABPP). Probes can either globally label reactive residues indiscriminately based on the nucleophilicity of the amino acid residues or mimic natural substrates, targeting classes of proteins. Given the dearth of small molecule therapeutics for RNA-binding proteins, we developed a purine-based probe scaffold to elucidate functional binding information.
Chapter 1 provides background information on RNA-binding proteins and ABPP with depth provided for cysteine residues. Chapter 2 will present the development of the purine-based probe scaffold and selectivity for nucleic-acid binding domains. Based on molecular recognition, we then develop a proteomics assay to identify RNA competition across the proteome. We finally identify and validate two novel RBPs. Chapter 3 elucidates a probe amenable Cys residue on selenocysteine elongation factor (eEFSec). Functional validation studies using a green-fluorescent protein (GFP) reporter highlight C442 as critical for selenocysteine (Sec) incorporation. Lastly, we screen nitrogen heterocycles for activity against this residue. Chapter 4 summarizes findings and discusses future directions for targeting RBPs.