Optical Tweezers to investigate at the single-molecule level the interaction of human Thymdylate Synthase with its consensus RNA

Human Thymidylate Synthase (hTS) is a homodimeric enzyme that plays a key role in DNA synthesis and a target for several anticancer drugs that bind its active site. However, the effectiveness of these drugs suffers from resistance effects, recently linked to the enzyme’s autoregulatory function. In performing this biological function, hTS binds its own messenger RNA (TSmRNA), sequestering the start codon for ribosomal translation, thus regulating its own concentration inside the cell. Thus, to design and develop more efficient chemotherapeutic drugs that preserve the autoregulatory function of hTS, a deeper understanding of both the structure of TSmRNA and its interaction with hTS is required. In this work, we use optical tweezers (OT) to study, at the single-molecule level, the structure of the binding site 1 of TSmRNA, by applying mechanical stimuli. OT allows the direct application of force to individual RNA molecules tethered between two micrometric beads, producing force-distance curves whose force jumps correspond to conformational changes of the molecule under study. First, experiments were realized in the absence of hTS, to characterize the RNA alone. The results indicate that the nucleotides involved in the unfolding/folding transitions are located in the apical region of the hairpin, corresponding to the last 23-nucleotide-long stem-loop, where the start codon AUG is present. This suggests that the most biologically relevant region of TSmRNA is also the one exhibiting the highest structural stability. Additionally, the free energy landscape of the molecule was reconstructed, calculating the kinetic rates relative to the unfolding/folding process and the free energy of formation of the molecule. This study has also represented a perfect benchmark for testing out-of-equilibrium statistical theories, like the two-state transition theory and the fluctuation theorems. Finally, pulling experiments on TSmRNA were also conducted in the presence of hTS, obtaining preliminary results that shed light on the mechanisms behind the formation of the TS:mRNA complex. Such experiments were performed with a newly designed microfluidic chamber, which allows obtaining more reliable experimental results.