# ATP/azobenzene-guanidinium self-assembly into fluorescent and multi-stimuli responsive supramolecular aggregates

Olivier Abodja, Nadia Touati, Mathieu Morel, Sergii Rudiuk, Damien Baigl

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Olivier Abodja, Nadia Touati, Mathieu Morel, Sergii Rudiuk, Damien Baigl. ATP/azobenzene-guanidinium self-assembly into fluorescent and multi-stimuli responsive supramolecular aggregates. Communications Chemistry, 2024, 7 (1), pp.142. 10.1038/s42004-024-01226-y. hal-04284864

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ATP/azobenzene-guanidinium self-assembly into fluorescent and
multi-stimuli responsive supramolecular aggregates

Olivier Abodja,¹ Nadia Touati,² Mathieu Morel,¹ Sergii Rudiuk,¹ Damien Baigl¹,.*

¹ PASTEUR, Department of Chemistry, École Normale Supérieure, PSL University,
Sorbonne Université, CNRS, 75005 Paris, France

²Chimie ParisTech, Université PSL, CNRS, Institut de Recherche de Chimie-Paris, PCMTH,
75005 Paris, France

* correspondence to: [damien.baigl@ens.psl.eu](mailto:damien.baigl@ens.psl.eu)

**Abstract**

Building stimuli-responsive supramolecular systems is a way for chemists to achieve spatio-
temporal control over complex systems as well as a promising strategy for applications ranging
from sensing to drug-delivery. For its large spectrum of biological and biomedical implications,
adenosine 5'-triphosphate (ATP) is a particularly interesting target for such a purpose but
photoresponsive ATP-based systems, such as photocaged compounds, have mainly been
relying on covalent modification of ATP. Here, we show that simply mixing unmodified ATP
with AzoDiGua, an azobenzene-guanidium compound with photodependent nucleotide binding
affinity, results in the spontaneous self-assembly of the two non-fluorescent compounds into
photoreversible, micrometer-sized and fluorescent aggregates. Obtained in water at room
temperature and physiological pH, these supramolecular structures are dynamic and respond to
several chemical, physical and biological stimuli, resulting in a multi-stimuli control of the
suspension turbidity and aggregate fluorescence. The presence of azobenzene allows a fast and
photoreversible control of their assembly upon cycles of UV (395/25 nm) and blue (480/30 nm)
irradiation of moderate power. ATP chelating properties to metal dications enable ion-triggered
disassembly and fluorescence control with valence-selectivity. Finally, the supramolecular
aggregates are disassembled by alkaline phosphatase in a few minutes at room temperature,
resulting in enzymatic control of fluorescence through ATP hydrolysis. These results highlight
the interest of using a photoswitchable nucleotide binding partner as a self-assembly brick to
build highly responsive supramolecular entities involving biologically relevant target molecules
without the need to covalently modify them.
Introduction

There is a general interest in developing stimuli-responsive systems due to their ability to
modulate their functions in response to endogenous signals or external stimulations¹,²,³. A
widely used strategy to convert a passive system into a responsive one, consists in tethering a
stimuli-responsive group, such as a photochrome, a pH-sensitive group or a redox species ⁴,⁵,⁶,
⁷,⁸ through adequate covalent modification. With the repertoire of known chemical reactions
and coupling methods, this powerful approach can lead to virtually any user-defined system
designed to respond to a desired stimulus. However, the necessity of covalently modifying the
entity to be stimulated can alter its properties. It also requires to adjust the modification
conditions for each system, making the approach case-specific. Stimulus-responsive self-
assembly of molecular bricks through non-covalent interactions appears as an interesting
alternative as it opens the possibility of dynamic actuation of inherently passive molecules
without needing to modify them. This approach has already been demonstrated relevant for a
large number of applications ranging from chemical sensors to drug-delivery systems⁹. The
resulting stimuli-responsive self-assembled systems are of particular interest when involving
biological molecules to dynamically control their functions¹⁰,¹¹,¹²,¹³. Among biologically active
molecules, nucleotides and analogues appear as particularly interesting targets for such a
purpose ¹⁴. For instance, adenosine 5'-triphosphate (ATP) is the universal energetic and
ubiquitous molecule driving biological processes such as cellular division and whose
concentration can correlate with pathological conditions, for instance in Parkinson's disease¹⁵.
Moreover, ATP bears negatively charged phosphates as well as an aromatic π-conjugated
adenine ring allowing, multiple electrostatic, π-π and hydrogen bonding interactions, thus
making it prone to be engaged in supramolecular self-assembled systems ¹⁶,¹⁷. Several stimuli-
responsive ATP-based supramolecular systems have thus been developed but for only a few
specific stimuli. One strategy consisted in exploiting ATP as an enzyme substrate to build
enzyme-responsive assemblies. This was achieved in particular using phosphatase to control
the fluorescence or chirality of the assemblies¹⁸,¹⁹,²⁰,²¹. Another approach explored the chelating
properties of ATP with metals to develop ion-responsive supramolecular structures but these
investigations were limited to small-sized complexes²², ²³,²⁴. Besides enzymes and metals, light
constitutes a highly desired trigger to control self-assemblies due to its precise spatiotemporal
control, tunability (wavelength, power) and remote character²⁵. Light-responsive ATP-based
systems to photochemically control the release of this nucleotide have been described but this
involved photocleavable bounds, therefore precluding any photo-reversible response²⁶. To our
knowledge, photoreversible supramolecular assemblies involving unmodified ATP have yet to
be demonstrated. To reach such a goal, azobenzene-containing molecules constitute promising
co-assembly partners as they have been successfully exploited in photocontrolled
supramolecular assemblies for their robustness and for a rapid and reversible *trans-cis*
photoisomerization²⁷. Cationic derivatives of azobenzene can be used in particular to
electrostatically interact with negatively charged mono- or polynucleotides, a process that can
be rendered photodependent through the polarity and/or geometric changes upon azobenzene
*trans/cis* isomerization²⁸. This was used for instance to photocontrol phenomena such as DNA
compaction²⁹,³⁰,³¹,³²,³³,³⁴,³⁵, co-acervation³⁶, DNA intercalation³⁷,³⁸ or G-quadruplex
formation³⁹. With the objective to build extended photoreversible ATP assemblies in water, and
thus limit the possibility of precipitation, we avoided classical azobenzene cationic
surfactants⁴⁰,⁴¹,⁴² and opted instead for a hydrophilic azobenzene partner, choosing guanidium
as the cationic group for its strong affinity for nucleotide phosphate groups, including those of
ATP⁴³,⁴⁴,⁴⁵. We thus used AzoDiGua, a symmetric diguanidinium azobenzene derivative and
photodependent DNA intercalator³⁷ which was shown to co-assemble with guanosine
monophosphate into photoswitchable and extended fluorescent crystals⁴⁶. In this work we co-
assembled AzoDiGua with ATP and observed the spontaneous formation in water of the first
photo-reversible micrometer-sized supramolecular assemblies containing ATP. The self-
assembled structures scattered light and exhibited fluorescence only in their assembled state, resulting in a reversible photoswitching of both turbidity and fluorescence. We also exploited the presence of ATP in the supramolecular structures to dynamically control their self-assembly by other stimuli, including metal ions and alkaline phosphatase (ALP) enzyme. With a combination of methods including optical microscopy, fluorescence spectroscopy, and electronic paramagnetic resonance (EPR), we characterized the reversibility, highly dynamic character and the switchable optical properties of these multi-stimuli responsive ATP/azobenzene-guanidinium assemblies of a new kind.

## Results and discussion

### Preparation of ATP/AzoDiGua supramolecular aggregates in solution

**Figure 1** shows the main components and concept of this work. ATP (Figure 1A, 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 AzoDiGua³⁷ which contains two guanidinium groups with a strong affinity for phosphates and an azobenzene group enabling both π-π interaction and photoswitchability (Figure 1A, 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 1B). 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 responsive⁴⁷. To evaluate the role of the phosphate groups in the ATP-AzoDiGua self-assembly process, we performed the same experiments using ATP analogs, and especially the monophosphate (adenosine monophosphate, AMP) and the diphosphate (adenosine diphosphate, ADP) derivatives (Figure S2). 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 S2A), in agreement with results previously obtained after a thermal annealing treatment⁴⁶. 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 S2B) than that with ATP (Figure S2C). 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 S3), 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 ¹⁶. 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 S4). 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 DNA³⁷ or with guanosine
monophosphate nucleotide⁴⁶. 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.
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Spontaneous assembly of an azobenzene diguanidinium nucleotide binder (AzoDiGua) and adenosine 5' triphosphate (ATP) into multi-stimuli responsive aggregates. A) Molecular structure and essential characteristics of AzoDiGua (left) and ATP (right). B) Scheme depicting the assembly set-up and resulting stimulus-responsive behaviour. C) Photographs of an ATP solution (1 mM, left), an AzoDiGua solution (1 mM, middle) and the resulting mixture after 10 minutes of incubation at room temperature (right). Each tube has a diameter 1 cm and contains 500 µL of solution. The enlarged image is a transmission</figcaption></figure>
microscopy image of the aggregates (scale bar: 20 µm). All experiments are done at room
temperature in 50 mM Tris HCl buffer solutions (pH = 7.4).

Fluorescence of the supramolecular assembly in solution

The presence of the AzoDiGua in the aggregates led us to explore whether fluorescent
properties could be acquired by these assemblies through the aggregation-induced fluorescence
mechanism of azobenzene⁴⁸,⁴⁹,⁵⁰,⁵¹,⁵²,⁵³. Using fluorescence spectroscopy, we found that the
ATP/AzoDiGua aggregates displayed a broad fluorescence excitation/emission with maxima at
400 nm and 650 nm for the excitation and emission, respectively (Figures 2A, S5). Notably,
neither ATP nor AzoDiGua alone was significantly absorbing for a fluorescence emission at
650 nm (Figure S5) nor fluorescent when excited at 400 nm (Figure 2A, S6). Moreover, the
same system prepared with ADP or AMP, instead of ATP, displayed a weak fluorescence for
ADP/AzoDiGua where small aggregates formed, and no detectable signal for AMP/AzoDiGua
where no assembly was observed (Figure S7). All these results show that the self-assembly
between ATP and AzoDiGua was instrumental in the emergence of the fluorescence from the
ATP/AzoDiGua suspension. Fluorescence microscopy (Figure 2B) evidenced that the
fluorescence emission was mainly localized at the position of the micrometer-sized
ATP/AzoDiGua aggregates (Figure 1C), which was thus attributed to the aggregation-induced
fluorescence of azobenzene within the supramolecular aggregates. Therefore, ATP and
AzoDiGua self-assembled to form fluorescent aggregates while their individual components,
AzoDiGua and ATP alone, were not intrinsically fluorescent in solution. We then studied how
different stimuli could dynamically control the assembly and therefore affect its aggregation-
induced fluorescence properties.
A)

<figure><img src="image_3.png" /></figure>

B)

<figure><img src="image_4.png" /></figure>

**Figure 2.** ATP/AzoDiGua aggregates are fluorescent upon assembly of non-fluorescent constituents. **A)** Fluorescence emission spectrum (excitation: 400 nm) of ATP alone (1 mM), AzoDiGua alone (1 mM), and the resulting mixture after 10 min of assembly. **B)** Fluorescence microscopy image (Exc: 475/20 nm; Em: 500-800 nm) of the supramolecular ATP/AzoDiGua aggregates shown in **Figure 1C right**. The scale bar is 20 µm. All experiments are done at room temperature in 50 mM Tris HCl buffer solutions (pH = 7.4).

**Photoresponse of ATP/AzoDiGua assembly**

Since the aggregates resulted from the co-assembly of AzoDiGua with ATP, we explored the challenge to realize the first photoreversible ATP-based supramolecular assemblies. Indeed, pure AzoDiGua is able to reversibly photoisomerize between *trans* and *cis* upon UV and blue light irradiation, respectively (Figure 3A)³⁷. To characterize the photoresponse of the supramolecular aggregates, we first prepared the ATP/AzoDiGua aggregates in the regular conditions ([ATP] = 1 mM, [AzoDiGua] = 1 mM, 10 min incubation at room temperature) using pure *trans*-AzoDiGua as a starting material and exposed them to successive irradiations by UV (395/25 nm, 26 mW·cm⁻²) for 3 min and blue (480/30 nm, 40 mW·cm⁻²) for 3 min. The
first UV application resulted in the progressive disassembly of the supramolecular aggregates until their complete disappearance in less than 1 min (Movie S2, Figure 3B *middle*). The subsequent blue irradiation led to the recovery of the assembly with a similar kinetics (Movie S2, Figure 3B *right*). This was accompanied by a photocontrol of the optical properties of the ATP/AzoDiGua suspension. The first UV irradiation led to the loss of the suspension turbidity due to the vanishing of the light-scattering entities and appearance of the characteristic color of the cis-AzoDiGua isomer enriching the solution (Figure 3B *middle*, Figure S8). Similarly, the subsequent exposure to blue irradiation induced a change of the optical aspect of the suspension which became turbid again, due to the reappearance of the micrometer-sized ATP/AzoDiGua aggregates (Figure 3B *right*). Note that, compared to the initial suspension composed of pure trans-AzoDiGua, the suspension after UV and blue irradiation was less turbid and the aggregates in smaller number and with a smaller size due to an incomplete trans-isomer enrichment upon blue irradiation in the photostationary state³⁷. This successive disassembly/reassembly of the ATP/AzoDiGua aggregates upon UV/blue irradiation was then successfully repeated for five complete consecutive cycles (Figure S9). It resulted in a dynamic and reversible photocontrol of the suspension fluorescence correlating with the assembly state, i.e., high intensity for the blue-induced assembly and low intensity after UV-induced disassembly, respectively, with only small fatigue appearing from the third cycle (Figure 3C). Again, the fluorescence after the first UV/blue cycle was less than in the initial solution (Figure S10) due to a lower fraction of trans-AzoDiGua in the photostationary state compared to the pure trans solution before irradiation. By exploiting the co-assembly between ATP and AzoDiGua, we could achieve both photoswitchable and aggregation-induced fluorescence of ATP/AzoDiGua aggregates leading to, the best of our knowledge, the first photoswitchable micro-meter sized ATP-based assembly.
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Photoreversible assembly of fluorescent ATP/AzoDiGua aggregates. **A)** Photoisomerization scheme of AzoDiGua upon UV and blue irradiation. **B)** Transmission microscopy images (main) and photographs (inset) of ATP/AzoDiGua suspension before (left) and after successive exposure to UV irradiation (395/25 nm, 26 mW·cm⁻²) for 3 min (middle) and blue irradiation (480/30 nm, 40 mW·cm⁻²) for 3 min (right). The scale bar is 20 µm. **C)**</figcaption></figure>
Fluorescence intensity (Exc: 400 nm; Em: 650 nm) of the ATP/AzoDiGua suspension after consecutive cycles of UV (395/25 nm, 26 mW·cm⁻²) for 3 min and blue irradiation (480/30 nm, 40 mW·cm⁻²) for 3 min. The initial suspension results from the self-assembly of assembly of ATP (1 mM) and AzoDiGua (1 mM) for 10 min. All experiments are done at room temperature in 50 mM Tris HCl buffer solutions (pH = 7.4).

## ATP/AzoDiGua assembly response to metal ions

To increase the stimulus-responsivity of the photoswitchable aggregates, we exploited the chelating properties of ATP to study the possibility to control this assembly with ions. Knowing the strong affinity of ATP with divalent metal ions²²,⁵⁴, our hypothesis was that the addition of such cations would lead to the disassembly of the aggregates and the decrease of their fluorescence (Figure 4A). To characterize this effect, we started from the preformed ATP/AzoDiGua aggregates and added different metal cations (Mⁿ⁺) to ensure a final molar ratio [Mⁿ⁺]/[ATP] = 2. Interestingly, it was found that the turbidity of the solution immediately disappeared for the tested divalent cations (Mg²⁺, Ca²⁺, and Cu²⁺) but remained for the monovalent ones (Na⁺, K⁺) (Figure 4B), showing that the ion-triggered disassembly was valence-selective. The color due to the presence of *trans*-AzoDiGua (Figure S8) remained upon ion-triggered disassembly as the response was mediated by ATP chelation and not by a change of AzoDiGua isomerization state. The mechanism of the metal-mediated disassembly of the aggregates was then further investigated in the case of Cu²⁺ using EPR spectroscopy, keeping all concentrations identical to that used for the assembly/disassembly experiments ([ATP] = 1 mM; [AzoDiGua] = 1 mM; [Cu²⁺] = 2 mM). The EPR signal of pure Cu²⁺ (2 mM) shifted to lower fields with appearance of a marked shouldering when it was mixed with ATP (1 mM) while ATP alone had no EPR signature for the same field range (Figure S11A). These effects,
in agreement with previous reports⁵⁵, are attributed to the interaction between Cu²⁺ and ATP upon chelation⁵⁶. On the contrary, the EPR signal of pure Cu²⁺ did not evolve upon addition of AzoDiGua (1 mM) showing the absence of interaction between these two cationic species (Figure S11B). Interestingly, AzoDiGua/ATP aggregates did not have any EPR signal but, after addition of Cu²⁺, a clear paramagnetic signal appeared and displayed the same features as the one of Cu²⁺/ATP mixture (Figure 4C). This new signal was thus attributed to the coordination of Cu²⁺ with ATP and shows that the disassembly of ATP/AzoDiGua aggregates upon Cu²⁺ addition is due to the competitive complexation between ATP and Cu²⁺. Optical microscopy observations of the ATP/AzoDiGua aggregates ([ATP] = 1 mM; [AzoDiGua] = 1 mM) exposed to increasing amounts of Cu²⁺ evidenced a gradual disassembly with an increase in [Cu²⁺] and showed that an excess of Cu²⁺ (2 mM) was necessary to ensure full and permanent disassembly (Figure S12). This ion-stimulated disassembly was then used to control the fluorescence of ATP/AzoDiGua co-assembly (Figure 4D, S13), showing either a way to chemically adjust the fluorescence of the system or an original method to titrate the presence of divalent cations (here, Cu²⁺) by fluorescence. After disassembly for a ratio [Cu²⁺]/[ATP]=2, further addition of ATP (3 mM) led to the re-assembly of ATP/AzoDiGua aggregates and appearance of suspension turbidity (Figure S14), emphasizing the reversibility of the supramolecular aggregates and the role of free ATP for the assembly to occur. By exploiting the chelating properties of ATP and its strong and specific affinity for divalent cations, we demonstrated that ATP-AzoDiGua aggregates were cation-responsive and valence-selective, offering the possibility not only to dynamically control both their assembly/disassembly behaviour and their fluorescence through a chemical stimulus but also to envision the principle of a divalent-specific ionic sensor.
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>Figure 4. ATP/AzoDiGua aggregates are cation-responsive and valence-selective. A)</figcaption></figure>

Scheme of metal ion-triggered ATP/AzoDiGua disassembly by ATP chelation with di and monovalent cations. B) Photographs of ATP/AzoDiGua suspensions before (left) and after (right) addition of different metal cations ($M^{n+}$) with a ratio $[M^{n+}]/[ATP] = 2$. C) Electron paramagnetic resonance (EPR) spectra at T=110 K of a mixture of $Cu(NO_3)_2$ (2 mM) and ATP (1 mM) (dashed line) and ATP/AzoDiGua suspension (solid line) before (purple) and after (green) adding $Cu(NO_3)_2$ at a ratio $[Cu^{2+}]/[ATP] = 2$. D) Fluorescence intensity (Exc: 400 nm; Em: 650 nm) of ATP/AzoDiGua suspension after addition of $Cu(NO_3)_2$ as a function of the ratio $[Cu^{2+}]/[ATP]$. For all experiments, the initial suspension results from the self-assembly of
assembly of ATP (1 mM) and AzoDiGua (1 mM) for 10 min. All experiments are done at room
temperature in 50 mM Tris HCl buffer solutions (pH = 7.4).

Enzymatic response of ATP/AzoDiGua assembly

We finally explored the ability to use an enzyme as a stimulus. Alkaline phosphatase (ALP)
was chosen as an enzymatic cleavage agent for its ability to catalytically hydrolyze ATP into
adenosine and three equivalents of monophosphate groups in very mild conditions. We
exploited this property and used ALP as a trigger to dynamically control ATP/AzoDiGua co-
assembly. Adding ALP to the preformed ATP/AzoDiGua aggregates ([ATP] = 1 mM,
[AzoDiGua] = 1 mM) led to their complete disassembly in 10 min (Figure S15). This
disassembly was accompanied by a loss of turbidity of the ATP/AzoDiGua solution after adding
the enzyme to the mixture (Figure 5A). When the same experiment was performed with a
previously denaturated ALP (treatment at 90 °C), the suspension turbidity remained and no
disassembly was observed (Figure S16), confirming that ALP-triggered disassembly was due
to ATP hydrolysis by the active enzyme. This enzyme-mediated disassembly was accompanied
by a gradual decrease of suspension fluorescence down to a minimal level corresponding to that
of free AzoDiGua after 10 min of reaction (Figure 5B), confirming the complete disassembly
and release of free AzoDiGua. These results thus demonstrated how both aggregation-induced
fluorescence of azobenzene and the characteristics of a self-assembled structure can be
dynamically controlled through the action of an active enzyme having as a substrate one of two
co-assembled partners.
<figure><img src="image_10.png" /><figcaption>**Figure 5.** Enzymatic control of the ATP/AzoDiGua assembly and fluorescence. A) Photographs of ATP/AzoDiGua suspension before (left) and after addition of alkaline phosphatase (ALP) for 10 min (right). B) Fluorescence intensity (Exc: 400 nm; Em: 650 nm) as a function of time for the ATP/AzoDiGua suspension without (green symbols) or with (red symbol) added ALP to ATP/AzoDiGua suspension at *t* = 0, or for AzoDiGua alone (1 mM) with added ALP at *t* = 0. For all experiments, the initial ATP/AzoDiGua suspension results from the self-assembly of assembly of ATP (1 mM) and AzoDiGua (1 mM) for 10 min. All experiments are done at room temperature in 50 mM Tris HCl buffer solutions (pH = 7.4).</figcaption></figure>

**Conclusion**

Adenosine 5'-triphosphate (ATP) is involved in many biological and biotechnological processes, making its dynamic actuation, and especially by light, a valuable challenge for a broad range of applications of fundamental and/or practical interest. Instead of the conventional approach involving a covalent modification to render it stimulus-responsive, we have described an original strategy where ATP is associated with a photosensitive nucleotide binder called AzoDiGua to form multi-stimuli responsive supramolecular structures through non-covalent interactions. AzoDiGua is a water-soluble azobenzene molecule with two guaninidium groups
as its extremity and known as a photosensitive DNA intercalator. Here, we exploited its
phototunable capacity to interact with nucleotides through π-π and electrostatic interaction to
build a new kind of photoresponsive co-assembly using ATP as a co-assembly partner. We
showed in particular that simply mixing AzoDiGua and ATP led to the spontaneous assembly
of the two partners into micrometer-sized aggregates in a few minutes in water. The interest of
such an approach is multifold. First, the approach allows to dynamically control the
assembly/release of ATP by light without having to modify it, and in mild conditions
(physiological pH, room temperature, ATP concentrations similar to intracellular one), making
the approach particularly robust, prone to generalization to other targets and opening interesting
perspectives for in situ non-invasive actuation. Second, the non-covalent assembly allows to
maintain the stimulus-responsiveness of the individual bricks in the supramolecular structure.
For instance, AzoDiGua remains photoswitchable and enables photoreversible
assembly/disassembly of the aggregates by short UV/blue irradiation pulses for several
consecutive cycles. Similarly, intrinsic chelation properties and enzymatic reactivity of ATP
could be exploited to dynamically trigger fast disassembly of the structures by the action of
metal cations or enzymes. Third, fluorescence appeared as an emerging property arising from
the self-assembly itself. Indeed, although ATP and AzoDiGua have only very weak fluorescent
properties when they are individually dispersed in solution, their co-assembly lead to
significantly fluorescent superstructures. Such aggregation-induced fluorescence of
azobenzene is a well-known phenomenon but is in many case obtained with pure azobenzene
compounds losing most of their photoswitchability in the aggregated state. Here, the co-
assembly with ATP allowed us not only to combine both aggregation-induced fluorescence and
photoswitchability in the same system, but also to realize multi-stimuli responsive fluorescence.
For instance, the fluorescence of the ATP/AzoDiGua supramolecular aggregates was
successfully and dynamically controlled by various physical (light), chemical (metal cations)
and biological (enzyme) stimuli. This not only further evidenced the dynamic control over the
self-assembly achieved by these different stimuli but also provides opportunities for the
development of sensing strategies for detecting ATP, analyzing the presence of metal cations
with valence-selectivity, or assessing enzymatic activity. Demonstrated here with two particular
bricks, ATP and AzoDiGua, we think this co-assembly strategy could be explored with other
nucleotide or DNA/RNA targets and/or other stimuli-responsive DNA intercalators to build
highly dynamic and multifunctional supramolecular structures in a particularly robust, user-
friendly and multi-purpose way.

Acknowledgement

We thank Laurent Binet (IMRCP, Chimie ParisTech, Université PSL, CNRS) for EPR support and discussion; Hélène Bertrand (LBM, Sorbonne Université, ENS-PSL, CNRS) for experimental support in the fluorescence spectroscopy measurements.

Funding

This work was supported by the European Research Council (ERC) (European Community's Seventh Framework Programme (FP7/2007-2013)/ERC Grant Agreement No. 258782) and the French National Research Agency ANR through contracts DYOR (ANR-18-CE06-0019) and SCDiag (ANR-21-CE18-0051).

**Conflict of interest**

The authors declare no conflict of interest.
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