Thymidylate synthase (TS) is a dimeric protein that represents the sole de novo pathway in human cells to deoxythymidilate (dTMP) synthesis, required for DNA synthesis and repair. Moreover, TS is also able to autoregulate its own expression inside cells, by interacting with its consensus mRNA sequence[1]: when TS binds to its mRNA, it inhibits the RNA translation, impeding the synthesis of further proteins. Thus, a good functioning of the TS catalytic process is essential for cell survival, and drugs targeting TS are widely used in chemotherapy against several types of cancer. However, treatments with classical TS inhibitors, such as fluorouracil, also affect the binding of the protein to its consensus mRNA, leading to overexpression of TS and the development of drug resistance [2]. Recently, an alternative approach has been proposed, based on the use of molecules able to bind to TS monomer/monomer interface, destabilizing the TS dimer. In this way, the catalytic activity typical of the dimeric enzyme is inhibited, whilst an increase in TS intracellular levels is not observed. Particularly this latter aspect requires further investigations, to better understand if the absence of protein overexpression is due to a modification in the TS/mRNA binding mechanism rather than a protein degradation[3]. Useful information on the TS-mRNA interaction in presence of the new inhibitors can be obtained by performing single-molecule experiments with optical tweezers (OT). OT represents a unique tool to investigate the biological world, thanks to their capability to manipulate one molecule at a time[4-6]. In this work, force spectroscopy experiments performed on the mRNA molecules will be presented, discussing the behavior of this molecule under the action of an external mechanical stimulus. The transition of the RNA molecule between its native and unfolded states has been characterized by means of pulling and releasing experiments, suggesting the cooperative unfolding/refolding of at least half of the whole nucleic chain. Furthermore, the kinetics of the RNA state transition, as well as the free energy landscape of the molecule will be discussed. Finally, preliminary results obtained both in presence of the mRNA and the TS will be presented, trying to clarify how the protein structure (dimeric/monomeric) and the presence of inhibitors could affect the mechanisms underlying the interaction of the enzyme with its consensus mRNA. [1] M.P. Costi et al, Curr. Med. Chem. 12, 2241, 2005[2] G.J. Peters, Cancer Drug Resist 1, 1, 2018[3] L. Costantino et al., eLife 11, e73862, 2022[4] A. Zaltron et al., Eur. Phys. J. Plus 135, 896, 2020[5] M. Rico-Pasto et al., PNAS 119, e2112382119, 2022[6] X. Viader-Godoy et al., Phys. Rev. X 11, 031037, 2021
