Small molecule guests influence the functional properties of supramolecular hydrogels. Molecular-level understanding of sol–gel compositions and structures is challenging due to the lack of long-range order and the inherently heterogeneous sol–gel interface. Here, we employ multinuclear (7Li, 11B, 23Na, 39K and 133Cs) gel-state magic-angle spinning (MAS) NMR spectroscopy to gain insight into the roles of alkali cations and borate anions in the gelation of guanosine (G)-quartets (G4). The MAS NMR spectra of alkali metal ions enabled the cations associated with G-quartets to be distinguished from the free cations. Combined 11B MAS NMR and modeling studies allowed cis and trans configurations of guanosine-borate (GB) diesters to be characterized, thus providing a complementary structural insight that indicates that the GB cis diester favours gelation. In addition, the multinuclear MAS NMR strategy is applied to understand the uptake process of biologically relevant small molecules into GB hydrogels. G4·K+ borate hydrogel can absorb up to 0.3 equivalent of cationic methylene blue (MB) without a significant disruption of the G4 fibrils that make up the gel, whereas the addition of over 0.3 equivalents of MB to the same gel leads to a gel-to-sol transition. By comparison, uptake of heterocyclic molecules such as adenine, cytosine and 1-methylthymine into G4·Na+ borate hydrogels leads to stable and clear gels while increasing the solubility of the nucleobases as compared to the solubility of the same compounds in water. G4·Na+ gel can uptake one equiv. of adenine with minimal disruption to the hydrogel framework, thus enhancing the adenine solubility up to an order of magnitude as compared to water. Multinuclear (1H, 11B and 23Na) MAS NMR analysis and vial inversion tests revealed that the nucleobases are embedded into pores of the solution phase rather than being closely interacting with the G4 fibrils that make up the gel phase. These results indicate that G4 hydrogels have potential applications as carrier systems for small molecule drugs.