|Development of a screening platform for the identification of macrocyclic splicing inhibitors
|Protein-RNA interactions are substance of research of the last decades and are yet to be fully explored. Interactions of RNA-binding proteins (RBPs) with RNA are found in a plethora of fundamental biological processes such as transcription, splicing, capping, polyadenylation, and translation. Alternative splicing (AS) is the major contributor to the high protein diversity in humans, with only ~30,000 genes present, and is regulated by RBPs such as spliceosomal subunits and alternative splicing factors. Aberrant function and expression of these factors disorganise splicing patterns, which can cause neurodegenerative diseases and cancer. Targeting alternative splicing factors to fight cancer has emerged as a novel promising therapeutic approach. For this purpose, two alternative splicing factors, SRSF1 and hnRNP A2B1, were first explored with the translational repression assay procedure (TRAP) to monitor the proteins’ interaction with RNA. The TRAP assay results delivered relative binding affinities, given in repression ratios, and were found to correlate with absolute binding affinities measured with the fluorescence polarisation (FP) assay. Binding motifs for RBPs are often found by cross-linking or pull-down approaches that are cost-intense and laborious. The work described here presents a novel approach using the TRAP assay in a high throughput format to screen for 10mer RNA consensus sequence for SRSF1 and hnRNP A2B1. The TRAP assay, in combination with the plasmid-encoded peptide library SICLOPPS, was used to screen for hexameric cyclic peptides as inhibitors for the RBPs. In a first approach, Sanger sequencing was used to sequence analysis and hit peptides were evaluated using the TRAP assay. Three hit peptides were synthesized chemically and evaluated by FP. In a second, optimised screening approach, Illumina sequencing was used for hit analysis, and the top six peptides were synthesised and evaluated by FP. Two promising candidate peptides for hnRNP A2B1 were found that are currently being analysed and verified as true inhibitors.
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|Fakultät für Chemie und Chemische Biologie
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