G-quadruplexes (G4s) are non-canonical four-stranded DNA helical structures formed through the stacking of four in-plane Hoogsteen-paired guanine bases.Conformational triggers of G4s have a huge potential in many areas, from nanotechnology to biomedicine. The aim of this thesis is to investigate the G4 conformational changes upon ligand complexation and by light activation. Firstly, a few small molecules were selected and tested as DNA ligands proving their capability to induce G4 secondary structure rearrangements and stabilisation. Among them, two photosensitive ligands were chosen: TMPyP4 porphyrin and photo-isomer pyridinium-decorated dithienylethene (DTE). Irradiation is a precise method in which timing, location, and strength of light can be easily controlled. Therefore, the use of photosensitive ligands could represent a molecular tailoring method for controlling changes in morphology and stability of G4s by non-invasive irradiation. Although several classes of drugs able to form complexes with G4s have been identified, the investigation of the G4-ligand interaction under different experimental conditions is still a challenge requiring the use of many techniques operating on different spatial and temporal scales.

Here, the results from a multi-technique approach combining structural and molecular information to get access to the G4s properties are reported. In-house techniques, such as UV-Visible absorption, circular dichroism and fluorescence spectroscopy, were combined with small-angle X-ray/neutron scattering methods and UV-resonant Raman spectroscopy available at large-scale facilities. To provide new insights from the experimental data, a multivariate singular value decomposition method was employed to extract quantitative information from complex datasets.

Overall, the proposed strategy allowed obtaining an experimental scheme to trigger conformational variations in the G4-drug complexes and to define the photo-physical parameters of the irradiation process (photo-reaction efficacy, irradiation time, photo-product stability) according to specific applications. One of the most important results of the work concerns the irradiation of the complex formed by the human G4 telomeric sequence AG3(TTAG3)3 (Tel22) andTMPyP4. At DNA concentration of tens of micromolar, irradiation with 430 nm partially destroys the Tel22 structure, possibly by oxidising the guanines. Interestingly, at a concentration one order of magnitude larger, porphyrin binding promotes Tel22 dimerisation and light irradiation increases dimer fraction.

This thesis is divided as follows: the first chapter is a brief introduction to the polymorphic world of G4s, focusing the attention on their structure, possible applications, and finally on the specific case of Tel22. The second chapter gives an overview of the techniques employed for the experiments: UV-Vis absorption, circular dichroism, UV resonant Raman, and fluorescence spectroscopy and smallangle X-ray/neutron scattering. An additional section is dedicated to the singular value decomposition method. The third chapter collects the results obtained for Tel22 alone. This sequence is highly polymorphic, so it is essential to define its conformational structural properties in different environmental conditions. The fourth chapter is about the conformational changes induced on the Tel22 structure by the complexation with a set of small molecules. The fifth chapter extends the analysis to photosensitive ligands (DTE and TMPyP4) interacting with Tel22.Both dark and light conditions were taken into account. The last chapter is focused on the Tel22 conformational changes induced by complexation with porphyrin TMPyP4 in the high-concentration regime. These conditions allowed the use of small-angle scattering techniques at the characterisation of the quaternary structure level. At the end, conclusions and perspectives are discussed.