G4s consist of planar G-quartets, formed by four guanine bases hydrogen-bonded in a Hoogsteen motif (Figure ), and these quartets can stack vertically to produce the full G4 structure. This stacking requires the presence of metal ions to stabilise the structure. Overall, G4s are therefore stabilised by the Hoogsteen hydrogen bonding to form quartets of bases and 𝜋 -𝜋 stacking interactions between G-quartets to form the G4 structure. There are also specific metal ion interactions (direct inner spheres coordination) with the O6 carbonyl oxygen of the guanine bases as well as nonspecific electrostatic ionic interactions. However, the relative importance and details of the combination of these multiple factors to the overall structure is not entirely clear. The structural analysis of nucleic acids has been predominantly approached through X-ray crystallography, NMR spectroscopy and cryo-EM, each providing complementary information. 2DIR spectroscopy is an optical analogue of 2D NMR spectroscopy, which measures the coupling between vibrational modes, which can occur electrically through space and additionally mechanically through bonds. The stretching frequencies of carbonyl groups in DNA bases are known to be sensitive to DNA conformation and conventional 2DIR methods have revealed that it is primarily strong coupling between carbonyl stretches in base pairs and between stacked bases that accounts for the frequency's observed sensitivity to structure. 2DIR spectroscopy has previously been used to explore DNA structural elements in a number of ways. For example, the analysis of DNA-ligand binding using 2DIR spectroscopy has also led to the development of an induced fit model for the binding of Hoechst33258 to the minor groove. 17 2DIR spectroscopy has also been used to study G4 DNA structures. In these studies, the dependence of the guanine carbonyl stretch frequency on the G-tract length was used to determine the preferred number of stacked G-quartets in extended G-tracts. Polarisation-dependent 2DIR spectroscopy was also used to distinguish between parallel and anti-parallel G4s.