# Reversible sequence-dependent DNA coacervation with an azobenzene intercalator

Yunzhe Li, Mathieu Morel, Damien Baigl, Sergii Rudiuk

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Yunzhe Li, Mathieu Morel, Damien Baigl, Sergii Rudiuk. Reversible sequence-dependent DNA coacervation with an azobenzene intercalator. 2025. hal-05368773

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# Reversible sequence-dependent DNA coacervation with an azobenzene intercalator

Yunzhe Li, Mathieu Morel, Damien Baigl and Sergii Rudiuk*

CPCV, UMR8228, Department of Chemistry, PSL University, Sorbonne Université, CNRS, Ecole Normale Supérieure, 75005 Paris, France

*Email: [sergii.rudiuk@ens.psl.eu](mailto:sergii.rudiuk@ens.psl.eu)

## Abstract

Based on electrostatic interactions and entropic gains from counterion release, DNA coacervates constitute an exciting example of associative assemblies due to high local DNA concentrations and possibility of their applications for drug and gene delivery. Because charge density is not affected by DNA sequence, resulting coacervates are intrinsically non-specific to the nucleobase composition of DNA. Here we report that using an azobenzene-based DNA binder, AzodiGua, able to intercalate between DNA base-pairs, can bring sequence-sensitivity to DNA coacervation. We show that different amounts of AzodiGua are necessary to induce coacervation as a function of DNA double- or single-stranded nature, as well as of its GC content. In addition, resulting coacervates were shown to be reversibly photosensitive via trans/cis isomerization of AzodiGua's azobenzene moiety, due to lower efficiency of the cis-isomer for DNA coacervation. The photocontrol could finally be reversed by adding α-cyclodextrin which selectively binds to trans-AzodiGua forming inclusion complex and hinders it from coacervation, without affecting the coacervation efficiency by the cis-isomer. UV-induced trans-to-cis isomerization of AzodiGua thus leads to dissolution of the coacervates in absence of α-cyclodextrin, and to their reformation in presence of α-cyclodextrin.
Introduction

Coacervation refers to associative liquid-liquid phase separation in polyelectrolyte solutions.<sup>1,2</sup> Typically observed in inter-polyelectrolyte complexes, similar process occurs upon electrostatic interactions of a polyelectrolyte with oppositely charged proteins or even small molecules.<sup>3</sup> Nucleic acid and/or protein coacervates are often considered as model systems for natural membrane-less organelles, such as nucleoli and stress granules, which compartmentalize biochemical reactions without needing lipid membrane,<sup>4</sup> and are able to affect cell behavior by sequestering cellular factors.<sup>5</sup> Coacervates have also been applied for immobilizing enzymatic cascades,<sup>6</sup> increasing local concentration of DNA-based biosensors,<sup>7</sup> performing non-enzymatic DNA ligation,<sup>8</sup> as well as for drug<sup>9,10</sup> and gene or mRNA vaccine delivery,<sup>11-14</sup> making reversible coacervation promising for efficient release of the loaded components. Various approaches have thus been used for reversible formation and dissolution of DNA coacervates, based on pH,<sup>14,15</sup> temperature<sup>15</sup> or redox<sup>14</sup> responsive coacervation agents. Due to its tunability and spatio-temporal resolution, photocontrol represents promising opportunity for actuation of DNA coacervates and liberation of DNA or other sequestered molecules on demand. One approach consists in introducing photoswitchable arylazopyrazole moiety directly into DNA before its coacervation with polylysine.<sup>22</sup> Another approach is based on using for DNA coacervation a positively charged photosensitive surfactant, AzoTAB.<sup>8,16,17</sup> At low DNA concentrations (µM) this molecule was used for photo-control of DNA compaction<sup>18,19</sup> and gene expression.<sup>20,21</sup> But, at higher DNA concentrations (mM), it was found to induce formation of complex DNA coacervate microdroplets, which were also shown to be photosensitive: the resulting coacervates could efficiently dissolve and re-form back upon UV and blue light illumination, respectively.<sup>16</sup>

Combination of photosensitivity with sequence-specificity can be of huge interest for sequestration and/or purification of DNA molecules by their sequence. A number of hybridization-based, and thus sequence-specific DNA microdroplets formed by liquid-liquid phase separation have recently been described.<sup>23-26</sup> However, even though sequence-specific electrostatically-based coacervation could be achieved for polypeptides<sup>27</sup> and proteins<sup>28,29</sup> by controlling the distribution of charged amino acids, due to homogeneous charge distribution along the DNA molecule, its complex coacervation is typically non-specific to DNA sequence and structure. The only example of sequence- and structure-specific DNA coacervation was reported by Matthew A. Tirell et al.<sup>30</sup> By investigating the interactions of DNA oligonucleotides with polylysines, it was found that while single-stranded (ss) DNAs were forming spherical coacervate microdroplets, double-stranded (ds) DNAs produced solid aggregates. In addition, ss-DNAs of different sequences were shown to have non-homogeneous effect on the phase behavior of their complexes with polylysine. This work clearly demonstrated differences in coacervation of single-stranded (ss) and double stranded (ds) DNA oligonucleotides with polylysines, but the observed effect of sequence was explained by formation of secondary structures through intra- or inter-oligonucleotide hybridization leading to the formation of ds-DNA fragments, rather than the effect of DNA sequence itself.

Our strategy to achieve sequence-specificity in complex coacervation of DNA consisted in implementing DNA intercalators, as inclusion of flat aromatic molecules between DNA base pairs is known to be sensitive to the sequence of DNA<sup>31,32</sup> due to differential interactions with AT and GC base pairs. Here we used photosensitive dicationic DNA intercalator, AzodiGua, for photosensitive and sequence-specific DNA coacervation. Previously we demonstrated that this molecule was not only able to intercalate into DNA double helix,<sup>33</sup> but also it could bind to DNA nucleotides through a combination of electrostatic interactions, π-π stacking and H-bonding.<sup>34,35</sup> Here we show for the first time that AzodiGua can induce liquid-liquid phase separation of DNA in structure- and sequence-specific manner. For this purpose we compare DNA/AzodiGua coacervation for single-stranded (ss) and double-stranded (ds) DNA of different sequences (%GC), and demonstrate two efficient approaches for making this process reversible: by photoinduced trans-cis isomerization of the azobenzene moiety, and by competitive host-guest binding of the intercalator to α-cyclodextrin.
## Results

Firstly, we characterized photo-induced trans-cis isomerization of AzodiGua (Figure 1A). In accordance to previous data, UV irradiation (365 nm, 0.35 mW·cm⁻²) of trans-AzodiGua (50 µM) led to gradual decrease of absorbance band at 360 nm corresponding to the trans-isomer and appearance of two new bands at 320 and 440 nm attributed to the cis-isomer (Figure 1B).³³ In these conditions photo-stationary state was achieved after 2 min of irradiation. We then investigated coacervate formation for 10 base pairs (bp) self-complementary DNA strand forming double-stranded (ds) DNA and containing 40% GC base pairs (abbreviated as ds-40%GC-DNA, Figure 1C) upon addition of trans-AzodiGua. Mixing clear solutions of ds-40%GC-DNA (500 µM in charge) and dicationic AzodiGua (300 µM) led in several seconds to the increase of turbidity (Figure 1D), and formation of microdroplets having 0.7 ± 0.1 µm diameter in the solution, as measured by optical microscopy (Figure 1E). To quantify the turbidity increase, absorbance of the samples was measured at 700 nm, the wavelength at which both DNA and AzodiGua do not absorb directly the light. Any increase in the absorbance at this wavelength is thus a result of light scattering by coacervate microdroplets. A typical turbidity curve for ds-40%GC-DNA as function of trans-AzodiGua concertation is shown in the Figure 1F. At low concentrations of AzodiGua the turbidity stayed at zero, upon further increase of [AzodiGua]*, turbidity increased rapidly before reaching a plateau. Because the height of the plateau was found to depend on DNA concentration and ionic strength of the solutions, we systematically normalized all turbidity curves by the maximum value corresponding to the plateau. From the turbidity curves, we then defined a characteristic AzodiGua concentration, noted [AzodiGua]*, and the corresponding characteristic AzodiGua/DNA charge ratio (R<sub>+/-</sub>*) at the onset of turbidity increase. These values were used for comparing the coacervation efficiency of different DNA with different coacervation agents. For 40%GC-DNA, the [AzodiGua]* was found to be 250 µM (R<sub>+/-</sub> = 1).
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Experimental system and characterization of DNA coacervates. A) Chemical structure and photoisomerization of AzodiGua (UV: 365 nm; blue: 440 nm). B) Absorbance spectra of trans-AzodiGua at increasing UV irradiation times (365 nm, 0.35 mW.cm⁻²). [AzodiGua] = 50 µM. C) Scheme and sequence of self-complementary 10 bp DNA having 40% GC base pairs (ds-40%GC-DNA). D) Photography of transparent solutions of ds-40%GC-DNA alone and AzodiGua alone (300 µM), and turbid mixture of both components (15 min after the mixing). Volume of the solutions is 400 µL, and the vials diameter is 11.6 mm. E) Optical transmission microscopy image of DNA coacervates from (D). F) Typical turbidity curve representing absorbance of ds-40%GC-DNA (500 µM) at 700 nm as function of AzodiGua concentration. [AzodiGua]* and R<sub>+/-</sub>* are defined as characteristic AzodiGua concentration and AzodiGua/DNA charge ratio at the onset of turbidity increase. All experiments are performed in 10 mM Tris/HCl, pH 7.4. [ds-40%GC-DNA] = 500 µM in charge.</figcaption></figure>

We then characterized the obtained DNA-AzodiGua coacervates. First, coacervation of ds-40%GC-DNA with trans-AzodiGua was studied at different ionic strengths of the solutions. We found that increasing [NaCl] from 0 to 300 mM (in 10mM Tris/HCl buffer) led to the increase of [AzodiGua]* from 250 to 890 µM (R<sub>+/-</sub>* from 1 to 3.6) (Figure 2A and Supplementary Figure S1), which confirms that DNA coacervation is based on electrostatic interactions between its phosphate groups and guanidinium functions of the AzodiGua. We then fixed the charge ratio R<sub>+/-</sub> to 1.2 and analyzed stability of the DNA-AzodiGua coacervates by measuring from transmission microscopy images the size of the observed microdroplets and its evolution with time. For 500 µM of DNA the size gradually increased during the first 2 h of incubation at room temperature from 0.7 ± 0.1 to 1.0 ± 0.2 µm (Figure 2B). When higher concentration of DNA was used (3 mM), the size of the microdroplets was twice bigger (1.6 ± 0.3 µm), and it also evolved during the 2 h of the observation up to 2.1 ± 0.2 µm. In the both cases,
steady-state size was achieved after around 2 h and no size evolution was observed after additional 2 h of incubation. To understand the origin of the stability of the microdroplets, we estimated whether they carried a residual charge by observing interaction of the microdroplets with surfactants. It was found that contrary to the positive DTAB, adding negatively charged SDS induced aggregation of the droplets (Supplementary Figure S2), which indirectly indicated net positive charge of DNA-AzodiGua coacervates, and agrees with the used charge ratio R<sub>+/-</sub> = 1.2.

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Physico-chemical properties of DNA-AzodiGua coacervates. A) Characteristic AzodiGua concentration ([AzodiGua]*) and AzodiGua/DNA charge ratio (R<sub>+/-</sub>) for 500 µM of ds-40%GC-DNA as function of NaCl concentration. B) Evolution of size measured by optical transmission microscopy for coacervate microdroplets formed with 500 µM or 3 mM (in phosphates) of ds-40%GC-DNA upon adding charge ratio R<sub>+/-</sub> = 1.2 of AzodiGua. C) Confocal fluorescence microscopy observation of coacervates formed by adding AzodiGua to a mixture of two different ds-40%GC-DNAs, one labeled with Cy3 and another one with Cy5. Left: green, Cy3 channel (λ<sub>Ex</sub> = 543 nm; λ<sub>Em</sub> = 582/35 nm); middle: red, Cy5 channel (λ<sub>Ex</sub> = 633 nm; λ<sub>Em</sub> = 726/143 nm), and right: composite image. D) Evolution with time of FRET efficiency for a mixture of ds-40%GC-DNAs labeled with Cy3 and Cy5 (250 µM each) with (red) and without (green) AzodiGua (R<sub>+/-</sub> = 1.2). Blue curve represents FRET efficiency for separately prepared and mixed (at t = 0) coacervates of ds-40%GC-DNAs-Cy3 and ds-40%GC'-DNAs-Cy5. The names of DNA are simplified to DNA-Cy3 and DNA-Cy5 respectively. E) FRET efficiency as function of temperature for a mixture of ds-40%GC-DNAs labeled with Cy3 and Cy5 (250 µM each) before (red) and after (blue) adding AzodiGua (R<sub>+/-</sub> = 1.2). The arrows indicate heating/cooling directions. For FRET measurement, λ<sub>Ex</sub> = 520 nm; λ<sub>Em</sub>(donor) = 550 nm, and λ<sub>Em</sub>(acceptor) = 682 nm. All experiments are performed in Tris/HCl buffer, pH 7.4.</figcaption></figure>

To analyze thermal stability of the coacervates, FRET measurements have been performed for
coacervates obtained by adding trans-AzodiGua to a mixture of two different ds-40%GC-DNAs labeled
with Cy3 and Cy5 respectively, abbreviated as ds-40%GC-DNAs-Cy3 and ds-40%GC'-DNAs-Cy5
(Supplementary Table S1). In confocal fluorescence microscopy of the resulting coacervates (Figure
2C and Supplementary Figure S3), both fluorophores were co-detected in all microdroplets, with a
FRET efficiency of 0.9 stable in time (Figure 2D, red curve), confirming the formation of coacervates
containing the both DNAs. By following FRET of this sample upon increasing the temperature, a steep
decrease of FRET efficiency from 0.9 to 0.2 was observed at around T = 80 °C (Figure 2E). Upon
cooling down this solution, FRET increased back to 0.9 at the same temperature showing a fully
reversible thermal dissolution of the coacervates at high temperatures. This shows that DNA/AzodiGua
coacervates are thus stable not only in time, but also in a wide temperature range.
We then investigated mixing of different coacervate microdroplets. For this purpose, coacervates of ds-40%GC-DNAs-Cy3 and ds-40%GC'-DNAs-Cy5 have been formed separately by adding R+/- = 1.2 of trans-AzodiGua to each DNA. Then, the both samples have been mixed and their interaction was followed by FRET measurements (Figure 2D, blue). Vary rapidly, during the time necessary to set up the measurement (1-2 min), FRET efficiency of the mixed sample increased to 0.78, and after 1 h achieved the value corresponding to the fully mixed coacervates. By confocal microscopy we however could observe not yet completely mixed sample, where each microdroplet already contained both DNAs, but the distribution of the DNAs within different microdroplets was still inhomogeneous (Supplementary Figure S4). This very rapid FRET increase occurred without any visible increase of the size of coacervates, indicating that the mixing occurred mainly by exchange of DNA molecules between the microdroplets, rather than by their physical fusion. To confirm that the microdroplets were in equilibrium with their surrounding phase, dilution experiment has been performed. Dilution of coacervates formed at R+/- = 1.2 with 10 mM Tris/HCl buffer led to steep decrease of the normalized turbidity, which achieved the value of 0 at dilution factor between 2 and 3, which was also confirmed by total disappearance of the microdroplets in optical microscopy (Supplementary Figure S5). This indicated that the system was not kinetically trapped, and change in the composition resulted in rapid response of the coacervates.

We then investigated whether AzodiGua would interact differently with ss- and ds- DNA of same length but different sequences (%GC) in absence and in presence of 100 mM NaCl. Turbidity curves systematically showed that i) more AzodiGua was necessary to induce coacervation at higher ionic strength, and ii) coacervation of single-stranded DNA occurred at lower concentrations of the intercalator independently on the ionic strength of the solution (Figure 3A,B, Supplementary Figures S6, S7). Interestingly, even though the onset of coacervation could be observed below the charge neutralization of DNA, stable coacervates are obtained at higher charge ratios and are charged positively (Supplementary Figure S2). In a control experiments we compared these results with DNA coacervation by monocharged trans-AzoTAB surfactant (Figure 3C,D), as mono-cationic azobenzenes were shown to not intercalate into the ds-DNA.<sup>33,36</sup> DNA coacervation with AzoTAB occurred at higher charge ratios, and in this case only slight difference was observed between the coacervation of ss- and ds-DNA, which can correspond to weaker charge density and smaller rigidity of the ss-DNA. The observed strong difference in coacervation of ss- and ds-DNA with AzodiGua thus originates from its intercalative properties. This indicates that, intercalation of AzodiGua into the dsDNA does not help the coacervation, and an excess of the intercalator is necessary to induce coacervation of ds-DNA when intercalation is possible.

We then compared the effect of DNA sequence (%GC) on the coacervation of ss- and ds-DNA. Figures 3A and 3B show characteristic R+/-* and [AzodiGua]* as function of %GC in 10 mM Tris/HCl. For ss-DNA, we observe only a slight decrease of [AzodiGua]* (and R+/-*)as function of %GC. Interestingly, independently on the salt concentration, bell-shaped curves were found as function of %GC for ds-DNA. For coacervation of ds-0%GC-DNA, the [AzodiGua]* was only slightly higher than that of ss-DNA, with increasing %GC it increased, reached a maximum at 40%GC, and then decreased back for ds-100%GC-DNA. In our knowledge these results represents the first example of sequence-dependent DNA coacervation. To understand the effect of sequence we measured binding constants of AzodiGua to the DNA of different %GC (Supplementary Figure S8), and found non-homogeneous evolution of the binding constant (K<sub>a</sub>) as function of %GC (Supplementary Figure S9). While practically the same K<sub>a</sub> was found for 0%GC and 100%GC ds-DNA (3.10x10<sup>3</sup> M<sup>-1</sup> and 3.13x10<sup>3</sup> M<sup>-1</sup> respectively), twice bigger binding constant was measured for the ds-40%GC-DNA (6.5x10<sup>3</sup> M<sup>-1</sup>). These results indicate a stronger binding of AzodiGua intercalator for mixed GC/AT pairs, correlating well with a weaker coacervation efficiency. In the control experiments with non-intercalating AzoTAB, no evident effect of DNA sequence was observed (Figures 3C,D). This further confirms that bell-shaped curves with ds-DNA originate from AzodiGua intercalation. All these results demonstrate that intercalation has the
effect of delaying the coacervation of ds-DNA, and higher concentrations of more efficient intercalator are necessary to induce DNA coacervation.

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Effect of sequence on DNA coacervation. Characteristic coacervation concentrations and characteristic charge ratios as function of %GC of DNA for ss- and ds-DNA for AzodiGua (A,B) and AzoTAB (C,D) in absence (A,C) and presence (B,D) of 100 mM NaCl. [DNA] = 500 µM in charge; 10 mM Tris/HCl, pH 7.4.</figcaption></figure>

We then studied if UV-induced photoisomerization of AzodiGua affected the coacervates. First, turbidity curves corresponding to coacervation of ds-40%GC-DNA with trans- and cis-AzodiGua were compared (Figure 4A). Interestingly, trans-cis isomerization of AzodiGua led to a shift of turbidity increase from [trans-AzodiGua]* = 250 to [cis-AzodiGua]* = 350 µM, showing that cis-isomer was less efficient to induce DNA coacervation. By fixing AzodiGua concentration at an intermediate value (300 µM), we applied on the coacervates five consecutive cycles of UV and blue light illumination (Figure 4B). Initial value of turbidity (i.e. absorbance at 700 nm) was normalized to 1. After only 5 s of UV illumination normalized turbidity rapidly dropped down to 0 showing full photo-dissolution of DNA coacervate microdroplets, which was confirmed by microscopy observations (Figure 4C). Blue light illumination of the resulting sample for 10 min induced slower reformation of the microdroplets accompanied with the increase of the normalized turbidity up to 0.24. Strong difference between turbidities of the initial trans-AzodiGua solution and samples obtained after blue-light induced cis-trans AzodiGua isomerization can be explained by the lower trans-isomer content in the photostationary state. Indeed, freshly prepared solutions of cationic azobenzene derivatives typically contain 100% of trans-isomer, whereas in the photostationary state under blue light illumination trans-AzodiGua content is usually about 75-80%.<sup>37</sup> This cycle was repeated 5 times without any loss of amplitude in the turbidity changes (Figures 4B,C). This demonstrates the photoreversible formation and dissolution of DNA coacervate microdroplets though AzodiGua photoisomerization.
<figure><img src="image_5.png" /><figcaption>**Figure 4. Reversible photocontrol of DNA coacervation.** A) Normalized turbidity as function of trans- and cis-AzodiGua concentrations. B) Five cycles of UV-induced dissolution and blue-light induced reformation of DNA coacervates. C) Transmission optical microscopy observation of DNA coacervates after consequence cycles of UV (10 s) and blue (10 min) irradiation cycles. All experiments are performed with 500 µM in charge of ds-40%GC-DNA in 10 mM Tris/HCl, pH 7.4. In (B) and (C), [AzodiGua] = 300 µM ($R_{+/-}$ = 1.2).</figcaption></figure>

Finally, we applied another approach to dissolve DNA coacervates based on using host-guest competitor for extraction of AzodiGua from the microdroplets. For this purpose, we used α-cyclodextrin (αCD) whose inclusion complexes with AzodiGua have been very recently characterized.³⁸ Indeed, AzodiGua efficiently binds to the αCD with binding constant $K_α = 1.96 \times 10^4$ M⁻¹ (Figure 5A and Supplementary Figure S10A), which is 3-fold stronger than its binding to the ds-40%GC-DNA. First, coacervates were formed in the usual conditions ($R_{+/-} = 1.2$ for 500 µM of ds-40%GC-DNA). Then, the effect of αCD addition was followed by optical microscopy (Figure 5B) and turbidity measurements (Figure 5C). Full dissolution of the coacervates occurred upon adding 60 µM of αCD, which corresponds to the ratio $R = [αCD]/[AzodiGua] = 0.4$. This indicates that efficient dissolving of the coacervates does not require complexation of all AzodiGua molecules, but only of their amount decreasing free AzodiGua concentration below its characteristic coacervation concentration [AzodiGua]*. Similar results have been observed for dissolution of DNA/AzoTAB coacervates (Figure 5C and Supplementary Figure S10B).
As αCD was shown to strongly bind only to trans-isomer of AzodiGua,³⁸ we applied this phenomenon to invert the photocontrol of coacervation. In this case, upon increasing AzodiGua concentration, conditions should be found at which trans-AzodiGua/αCD inclusion complex would not coacervate DNA, but upon UV-induced trans-cis isomerization, sufficient for coacervation amount of cis-AzodiGua would be expelled from the αCD. Such conditions have been found for [AzodiGua] = 1200 µM. In these conditions, adding 4800 µM of αCD induced total dissolution of the coacervates, whereas consequent UV illumination led to re-formation of the coacervates (Figures 5E and 5F). Following blue light illumination then only partially redissolved the coacervates. Even though this process shows limited reversibility, in our knowledge this result represents the first example of inverted photocontrol of azobenzenes through selective sequestration of trans-isomer by cyclodextrins.

<figure><img src="image_6.png" /><figcaption>**Figure 5.** Dissolution and inverted photocontrol of coacervates through host-guest complexation with α-cyclodextrin. A) Scheme of formation of trans-AzodiGua/αCD inclusion complex. B) Optical microscopy observation of dissolution of ds-40%GC-DNA/AzodiGua coacervates (R+/- = 1.2 for 500 µM in charge of DNA) upon addition of αCD. C) Normalized turbidity of ds-40%GC-DNA coacervates formed with AzodiGua and AzoTAB as function of αCD concentration, represented by the ratio R = [αCD]/[AzoX]. [ds-40%GC-DNA] = 500 µM in charge; R+/- = 1.2 for both AzodiGua and AzoTAB. D) Scheme showing release of cis-AzodiGua from αCD upon photoisomerization. E,F) Optical microscopy (E) and turbidity measurements (F) for inverted photocontrol of DNA coacervation with AzodiGua/αCD complex. [ds-40%GC-DNA] = 500 µM in charge; [AzodiGua] = 1200 µM; [αCD] = 4800 µM. UV represents 20 min illumination at 365 nm, and blue corresponds to 20 min illumination at 440 nm. All experiments are performed in 10 mM Tris/HCl buffer, pH 7.4.</figcaption></figure>
In conclusion, here we described a novel approach for DNA coacervation based on photosensitive DNA binder AzodiGua. Due to its specific intercalative properties, differences were found in coacervation not only between single-stranded and double-stranded DNA, but also between double-stranded DNA having different nucleobase compositions (%GC). This finding opens the path toward sequence-dependent DNA coacervation by specific DNA binding molecules: not only intercalators, but also minor and major DNA grove binders.

## Acknowledgments

This work was supported by French National Research Agency (ANR) contracts ActiveGEL (ANR-18-CE07-0001) to S.R., DyNAmyl (ANR-22-CE06-0009) to S.R. and DYOR (ANR-18-CE06-0019) to D.B., by the European Research council ERC under the European Unions "HORIZON EUROPE Research and Innovation Programme (Grant Agreement No 101096956)" to D.B., and by China Scholarship Council (CSC) fellowship to Y.L. (202008420235).
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