Autoregulatory function of Thymidylate Synthase: a single-molecule investigation using optical tweezers

Thymidylate Synthase (TS) is a dimeric enzyme that represents the sole de novo source of thymidine in human cells. Besides this catalytic role, TS also performs an autoregulatory function by binding its own messenger RNA (TSmRNA), thereby blocking ribosome interaction and the translation of further TS proteins. Although TS is a widely employed target in the treatment of solid cancers, as the ovarian and pancreatic ones, current chemotherapeutic drugs targeting TS disrupt its autoregulatory mechanism, leading to chemoresistance effects. The present thesis proposes to investigate, at the single-molecule level, the interaction between TS and TSmRNA, to elucidate the structural and kinetic mechanisms underlying TS autoregulation, poorly characterized in the past. Indeed, the detailed understanding of the autoregulatory function of TS is essential for the development of more efficient chemotherapeutic drugs. An optical tweezers instrument is employed to perform single-molecule force-spectroscopy measurements by manipulating individual TSmRNA molecules and monitoring in real-time the formation of the TS:TSmRNA complex. A combination of kinetic barrier-crossing theories, polymer elasticity models and out-of-equilibrium dissipative statistical approaches are used to analyse the data and characterize the kinetics and free energy landscape of the TS:TSmRNA complex. The findings of this study, therefore, are expected to advance the rational design of therapies capable of inhibiting TS catalytic activity while preserving its autoregulatory function, opening new avenues in the fight against chemoresistance in solid tumours.