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The interaction between proteins and RNA has gained increased attention as a key regulatory mechanism of RNA translation beyond conventional postulated mechanisms. It is also involved in various pathologies, including cancer and neurodegenerative disorders. In this thesis, the interactions between the RNA-binding protein Musashi-1 (MSI1) and both linear and hairpin-like RNA strands have been investigated using time-resolved and structural technologies, such as SPR biosensors, LigandTracer, and NMR.

Initially, the interactions of the two RNA recognition motifs (RRM1 and RRM2) in MSI1 were characterized separately. Both motifs exhibited high affinity for the UAG motif, with no significant difference between linear and hairpin-like RNA. These interactions followed a 1:1 binding mechanism, with relatively fast, but well-defined association and dissociation rate constants. Next, the interaction of the N-terminal region of MSI1, containing both RRMs, was analyzed, revealing a bivalent binding mechanism. Commercial software was not suitable for distinguishing the fast monovalent and slow bivalent interactions. Therefore, a novel method was developed to quantify kinetic rate constants and binding affinities.

To explore the determinants for the specificity of the interaction, RNA mutants and protein variants were designed to shift RRM2 recognition from the UAG to the CAG motifs. Comprehensive characterization confirmed the impact of these mutations and RNA structural modifications on the interaction.

Additionally, to examine MSI1 regulatory role in synthetic biology, a post-transcriptionally controlled circuit was engineered in E. coli using MSI1 binding to RNA strands. This system, developed on solid media, utilized sfGFP fluorescence as a reporter. Data analysis using the Gompertz growth model correlated well with previously published data on bacteria in suspension. The regulatory effect of oleic acid, a potential MSI1 allosteric inhibitor, was confirmed using this assay. Similarly, MSI1 inhibition effects were assessed in HCT-116 colorectal cancer cells, revealing that both oleic acid and luteolin impacted cell proliferation and CD44v6 receptor expression.

In conclusion, this work has resulted in novel methods and insights for studying and understanding the kinetics and mechanisms of protein-RNA interactions.