High-Throughput Particle Display Screening of RNA-Protein Interactions ; Prime Editor-Mediated Programmable Insertion of UAAs into Endogenous Proteins

Abstract

High-Throughput Particle Display Screening of RNA-Protein Interactions RNA-protein binding interactions have essential roles in many biological processes including transcriptional and translational control; thus, it is important to quantify the binding affinities of these biological complexes through functional binding assays. Although conventional binding assays have provided significant insight to these dynamic networks, they generally provide a relatively low throughput for a limited number of samples. To overcome the limitations of these conventional binding assays to study RNA-protein binding interactions, we propose to develop an in vitro, high-throughput particle display-based for RNA aptamer screening of RNA-protein complexes for the subsequent identification and characterization of novel RNA aptamers that influence protein binding. With this technique, we will be able to profile large numbers of binding events based on binding-induced fluorescence-enhancement for a more holistic understanding of the corresponding RNA-protein network. So far, we have confirmed that this particle display-based technique can be used to estimate the binding affinity of the well-characterized MS2-MCP model system, and plan to advance this technique to screen a library of MS2 variants for mutational analysis. Prime Editor-Mediated Programmable Insertion of UAAs into Endogenous Proteins The introduction of unnatural amino acids (UAAs) to endogenous cell surface proteins for site-specific bioconjugation reactions allows for the incorporation of clickable, fluorescent handles in vivo; however, the transient expression of proteins harboring UAAs is limited by its transfection efficiency. Thus, we propose to employ prime editors and tRNA/aminoacyl-tRNA synthetase technologies to introduce an UAA to endogenous proteins for downstream bioconjugation applications. Briefly, we propose to stably incorporate a stop codon into mammalian cells by prime editing which will be confirmed with a reporter system, such that this stop codon can mediate the introduction of an UAA through the associated tRNA/aminoacyl-tRNA synthetase technology. By permanently introducing a bioorthogonal, clickable handle onto an endogenous protein, its cellular signaling and localization patters can be monitored in vivo for further classification of the behaviors of these proteins. So far, we identified a promising fluorescent reporter construct to validate the introduction of a stop codon into the mammalian genome by prime editing.