Optical tweezers to investigate at the single-molecule level the interaction of thymidylate synthase with its consensus mRNA

The Thymidylate Synthase (TS) is an enzyme widely employed as a target in the treatment of various solid cancers. Nevertheless, the clinical effectiveness of the current drugs is limited by some drawbacks, which are mainly related to the interaction of TS with its consensus RNA (TSmRNA). The most employed drugs targeting TS negatively affect its autoregulatory function, which consists of the capability of TS to bind the mRNA and inhibit the translation process, thus controlling its own concentration inside the cell. Recently, new approaches have been proposed to target TS while preserving its autoregulatory function. However, the design and development or more efficient chemotherapeutic drugs necessarily require an in-depth knowledge of both the structure of TSmRNA and the binding mechanisms involved in its interaction with TS. In this work, for the first time, we used optical tweezers to investigate at the single-molecule level the structure of the binding site 1 of TSmRNA, by applying an external mechanical stimulus. We performed out-of-equilibrium pulling experiments, where each individual molecule was stretched and released several times, while monitoring its transitions among its folded and unfolded forms. The Worm-like Chain model was employed to analyze the force-distance curves, deriving information on the number of nucleotides involved in the conformational changes of TSmRNA and its stability.