Preparation of ATP/AzoDiGua supramolecular aggregates in solution Figure shows the main components and concept of this work. ATP (Figure , left) was used as a biologically active brick to be self-assembled. To ensure the formation of extended and light-responsive assembly, ATP was co-assembled with the photosensitive DNA intercalator AzoDiGua which contains two guanidinium groups with a strong affinity for phosphates and an azobenzene group enabling both π-π interaction and photoswitchability (Figure , right). We hypothesized that mixing ion-and enzyme-sensitive ATP with photosensitive AzoDiGua would lead to the spontaneous formation of multi-stimuli responsive assemblies in water at room temperature (Figure ). The direct equimolar mixture of the two components ([ATP] = [AzoDiGua] = 1 mM) at physiological pH (7.4) and room temperature led to the immediate formation of a turbid solution. Optical microscopy observations revealed the presence of micrometer-sized entities that progressively aggregated with each other over time (Figures 1C, S1, Movie S1). After 10 min, the aggregates reached an average size of around 20 µm in average. Assemblies of similar size and morphology were recently reported when ATP was mixed with oppositely charged ammonium-or guanidinium-rich polycations, highlighting the interest of exploiting electrostatic interactions but the resulting structures were not responsive . To evaluate the role of the phosphate groups in the ATP-AzoDiGua selfassembly process, we performed the same experiments using ATP analogs, and especially the monophosphate (adenosine monophosphate, AMP) and the diphosphate (adenosine diphosphate, ADP) derivatives (Figure ). After the same self-assembly time (10 min), a markedly different behaviour was observed between AMP, ADP and ATP. With AMP, the solution remained transparent and no aggregate could be detected by optical microscopy (Figure ), in agreement with results previously obtained after a thermal annealing treatment . In contrast, both ADP and ATP led to the formation of supramolecular aggregates but those obtained with ADP were of much smaller size (8 ± 1 µm) and led to a less turbid solution (Figure ) than that with ATP (Figure ). This evolution not only emphasizes the crucial role of the phosphate groups for the supramolecular self-assembly to occur but provides also a way to tune the aggregate characteristics by the number of phosphate per AzoDiGua nucleotide partner. When using ATP at an acidic pH (2.0 instead of 7.4), the ATP/AzoDiGua mixture solution remained transparent and no extended supramolecular structures could be detected by optical microscopy (Figure ), which was attributed to a lower ionization degree of ATP (-2 ionization state at acidic pH), its possible hydrolysis to ADP at acidic pH or a combination of both effects . All these results show the important role of the electrostatic interactions between the negatively charged phosphate groups of ATP and the cationic guanidinium moieties of AzoDiGua. To have further insights into the molecular interactions between AzoDiGua and ATP at a low concentration where microscopic aggregation did not occur, a spectroscopic titration of AzoDiGua by ATP was performed. We observed a hypochromic effect on the π-π * transition of AzoDiGua at 360 nm with increasing amounts of ATP (Figure ). This effect is in agreement with the establishment of π-π interactions between ATP and AzoDiGua, which are probably involved in the formation of the supramolecular aggregates at higher ATP and AzoDiGua concentrations. ATP is thus shown as a new binding partner to AzoDiGua, a DNA intercalant known so far for its propensity to form π-π and electrsotatic interactions within double-stranded DNA or with guanosine monophosphate nucleotide . In summary, ATP and AzoDiGua spontaneously self-assembled at room temperature and physiological pH into supramolecular aggregates through combined effects of electrostatic interactions and π-π stacking. These aggregates reached a size of a few micrometers in only a few minutes. To characterize their properties, including their reversibility and dynamic character, we fixed their composition ([ATP] = [AzoDiGua] = 1 mM) and assembly time to 10 min before studying their fluorescence and response to the application of different physical, chemical and biological stimuli.