# In silico design of a solution-gated graphene transistor sensor for the efficient detection of guanine quadruplexes

Aurianne Rainot, Luisa D'anna, Alessio Terenzi, Raphael Rouget, Stéphanie Grandemange, Benoit Piro, Giampaolo Barone, Florent Barbault, Antonio Monari

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Aurianne Rainot, Luisa D'anna, Alessio Terenzi, Raphael Rouget, Stéphanie Grandemange, et al. In silico design of a solution-gated graphene transistor sensor for the efficient detection of guanine quadruplexes. Journal of Physical Chemistry Letters, 2024, 15 (43), pp.10881-10887. 10.1021/acs.jpclett.4c02796. hal-04761163

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# In Silico Design of a Solution-Gated Graphene Transistor Sensor for the Efficient Detection of Guanine Quadruplexes

Aurianne Rainot, Luisa D'Anna, Alessio Terenzi, Raphael Rouget, Stéphanie Grandemange, Benoit Piro, Giampaolo Barone, Florent Barbault,* and Antonio Monari*

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Cite This: J. Phys. Chem. Lett. 2024, 15, 10881–10887

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Supporting Information

**ABSTRACT:** Guanine quadruplexes (G4s) are nucleic acid structures present in diverse regions of the genome, such as telomeres and transcription initiators. Recently, the different biological roles of G4s have been evidenced as well as their role as biomarkers for tumors or viral infections. However, the fast and efficient detection of G4s in complex matrices remains elusive. In this contribution, by using long-scale molecular dynamics simulations, we propose the design of a biosensor based on organic field-effect transistors recognizing G4s. In particular, we show that the interaction of the G4s with the biosensor is translated into a change in the charge density profile, which correlates with the electrical transduction of the signal, thus allowing the detection of the nucleic acid structure. We also provide rules of thumb for the optimization of the design of the device and more generally for the integration of computationally driven design approaches.

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Guanine quadruplexes (G4s) are nucleic acid structures formed in guanine rich regions of nucleic acid sequences, both DNA and RNA. G4s are formed by the superposition of π-stacked quartets of guanine, which are locked by Hoogsten hydrogen bonds.¹ Although most of the known G4 structures derive from the folding of a single-stranded oligonucleotide, multiple nucleic acids may also couple to produce intermolecular G4 arrangements.² Interestingly, G4s present a rather complicated and polymorphic behavior³ characterized by the coexistence of a rigid core and flexible lateral loops.⁴,⁵ The G4 core is also stabilized by the inclusion of monovalent cations, usually K⁺ or Na⁺, in the central channel of the tetrads to counterbalance the locally high electronic density. Furthermore, G4s may also exhibit a subtle conformational equilibrium, leading to the coexistence of parallel, hybrid, and antiparallel folding, depending on the relative orientations of the oligonucleotide strands. The folding of a G4 may also be influenced and even dictated by external factors, such as crowding agents or salt buffer. The crucial biological role of G4s has recently been underlined. Indeed, G4s are present, for example, in the telomeric regions of the DNA,⁶⁻⁸ where they exert a protective role and may inhibit telomerase,⁹⁻¹¹ hampering the emergence of the immortality phenotype, which is a hallmark of cancer cells. Moreover, G4s are also present in noncoding regions of the genome as consensus-regions for transcription factors.¹²⁻¹⁶ This involves crucial oncogene promoters such as KRAS,¹⁷,¹⁸ cMYC,¹⁹ hRAS,²⁰ and KIT.²¹,²² The stabilization of the G4 structure has been linked to the general suppression of the gene expression and/or translation,¹⁴,¹⁵ leading to interesting therapeutic strategies. In some cases the presence of G4 structures can instead induce

gene expression/translation. For example, an RNA G4 within the 5'-UTR of VEGF is key for internal ribosomal entry site (IRES)-mediated translation initiation.²³,²⁴ G4s have also been identified in the genome of both bacteria²⁵,²⁶ and viruses,²⁷,²⁸ including coronaviruses, such as SARS-CoV-2,²⁹⁻³¹ and flaviviruses.³² The possible formation of G4s in RNA sequences has, indeed, been confirmed for eukaryotic, prokaryotic, and viral organisms.³³,³⁴

Recently, the presence of G4s in circulating DNA has also been considered as a potential cancer biomarker, sparking considerable interest toward their accurate detection.³⁵,³⁶ Furthermore, due to the presence of G4s in viruses and bacteria, their rapid detection would also be beneficial for alternative rapid and large-scale screening campaigns in the case of epidemic outbreaks. While some specific G4 binders,³⁷⁻⁴² including DARPin peptides,⁴³,⁴⁴ have been proposed to this scope, at the moment no straightforward and fast detection of G4 structures exists. The G4 conformation can be relatively easily identified in aqueous solution by circular dichroism techniques,⁴⁵,⁴⁶ although its quantification in complex matrices, such as blood, is problematic and is usually based on DNA or RNA extraction and sequencing or the exploitation of bioinformatics tools identifying putative

Received: September 25, 2024

Revised: October 17, 2024

Accepted: October 21, 2024

Published: October 23, 2024

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>**Figure 1.** Structure of TMPyP4 (A), anthracene (B), pyrene (C), and coronene (D).</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 2.** (A) Free energy profile for the dissociation of the π-stacked interaction with the graphene layer. The precise definition of the collective variable is given in the SI. (B) Snapshots showing the equilibrium bound-state (cyan) and the bulk unbound-state (salmon) for TMPyP4.</figcaption></figure>

quadruplex forming sequences. Clearly this situation precludes large-scale screening for the presence of G4s and is not suited to discriminating the competitive folding landscapes.

In this contribution, we propose an original strategy for G4 detection based on the use of solution-gated graphene transistor sensors (SGGTs).$^{47-50}$ The latter are field-effect transistor devices based on graphene as an active layer and gated through a liquid electrolyte. In short, graphene is a highly conductive semimetal into which charge carriers (electrons and holes) have extremely high mobility. However, being a 2D material, graphene's electrical properties are highly sensitive to its chemical and electrostatic environment. If a voltage difference is imposed between the graphene layer and an electrode dipping in the electrolyte (termed a "gate"), the electrolyte/graphene interface is polarized; that is, ions are redistributed at this interface. For example, if anions (cations) are overrepresented at the liquid side of this interface, holes (electrons) will be overrepresented within graphene, which increases its conductivity, i.e., the current that flows through it. The principle is the same if charged molecules (such as polyanionic RNA) are immobilized on the graphene layer: the hole density within graphene depends on the distribution of charges and is linked to the conformation (extended, folded) of the RNA strand (i.e., how far it extends from the interface). Transistors being amplifiers by nature, small changes in ion distribution are translated into a large change in the measured current, which makes them excellent sensors. For such kinds of

applications, the graphene interface is sensitized with a molecular probe, which is usually π-stacked with the graphene layer, since π-stacking immobilization, rather than covalent binding, avoids degrading the graphene's conductivity. Upon interaction of the surface-bound probe with the analyte, here G4, a significant modification of the local charge density and of the electrostatic potential is translated into an electrical response of the sensor, generally a change in voltage or in current. Despite its apparent simplicity, the precise design of SGGTs, especially aimed at the selective detection of G4s, is challenging.

We propose a strategy based on the sensitization of the graphene layer with a synthetic probe constituted by a known G4 binder (such as TMPyP4,$^{51-54}$ Figure 1) and a conjugated polycyclic aromatic unit, which should interact specifically with graphene. The two moieties are bridged by a linker, inserted by click chemistry approaches. To optimize the rational design of the SGGTs, we have performed long-time molecular dynamics (MD) simulations complemented with enhanced sampling and free energy methods. We show that the proposed probe is able to persistently anchor to the graphene layer, to interact with G4, and to affect the charge density profile in the vicinity of the graphene/electrolyte interface, which should lead to an enhanced voltage and current response through the transistor.

The MD simulations have been performed using, consistently, the Amber software,$^{55}$ as well as the generalized amber force field for the organic probes, while amberff99 including
<figure><img src="image_11.png" /><figcaption>**Figure 3.** (A) The chosen TMPy4P4–Coronene dimer (Cor/TP4) along with snapshots from the MD trajectories in the presence of graphene identifying two binding modes with TMPy4P4 perpendicular (B) and parallel (C) to the surface.</figcaption></figure>

the DNA bsc1⁵⁶,⁵⁷ corrections has been used for the nucleic acid. All simulations have been performed in the isothermal and isobaric ensemble, while umbrella sampling has been used to obtain potential of mean force (PMF) and free energy profiles. Extended details of the computational strategy employed, as well as the precise definition of the collective variable used, are provided in the **Supporting Information** (SI).

Having chosen to rely on a SGGTs approach, the first challenge is to find a hybrid probe that selectively interacts with the graphene sheet, leaving the TMPyP4 available for locking the G4. To this aim, we have performed equilibrium and enhanced sampling MD simulations of a graphene sheet interacting with TMPyP4, as well as with pyrene, anthracene, and coronene (Figure 1). We also determined the free energy profile related to the dissociation of the π-stacked ligand and the graphene layer. The main results are collected in Figure 2. The chosen collective variable has been described by considering the projection over the axis perpendicular to the graphene sheet of the distance between the center of mass of the ligand and four carbon atoms placed at the four corners of the graphene layer. More details are provided in the SI. We may see that all of the considered compounds lead to stable stacking aggregates with the graphene layer. This is not surprising, considering that all of the considered systems may be referred to as conjugated polyaromatic systems. However, one could expect that the positive charges of TMPyP4 would hamper its interaction with the hydrophobic graphene layer. Yet, the large π-conjugation of TMPyP4 seems to dominate over electrostatic repulsion, and indeed, its aggregate with graphene is more stable than those with pyrene and anthracene. However, coronene binds stronger to graphene, with the free energy preference for the latter over TMPyP4 amounting to about 10 kcal/mol. Therefore, we identified a strong correlation between the number of aromatic rings and the stability of the aggregate with graphene. Interestingly, a rather large flat plateau showing some moderate oscillations at short distances is observed in the free energy profile.

This is because the aromatic ligand is sliding on the graphene surface and explores different quasi-degenerate

configurations. It is noteworthy that, regardless of the ligand interacting with graphene, the dissociation from the surface does not show any roughness in the free energy profile but rather a steady increase until reaching a plateau. We believe that this fact correlates with rigid and planar ligands presenting extended aromaticity, which do not require conformational changes such as dihedral angle torsions to adsorb onto the graphene surface.

The difference in the binding free energy obtained for coronene and TMPyP4 clearly suggests that hybrid dimeric probes composed of these two units will preferentially interact with the coronene moiety on the graphene surface. This encouraging result led us to propose (Figure 3A) a probe bridging TMPyP4 to a coronene unit via a triazole bridge (Cor/TP4).

As a proof of concept, we have run equilibrium MD simulations of the Cor/TP4 unit in the presence of a graphene sheet (Figure 3B,C). In agreement with the results of the free energy profiles for the individual constituents, we have observed that the hybrid dimer is preferentially interacting with graphene via its coronene unit.

However, we may also observe that in the case of one replica, the TMPyP4 unit has developed persistent stacking interactions with the graphene, leading to a globally planar arrangement of the dimer. This feature, which may lead to a loss of sensor performance, is most probably due to insufficient stiffness or hydrophilicity of the linker. Thus, we also investigated the behavior of a model parallel G4, namely, the cMYC proto-oncogene promoter (pdb code 1XAV)⁵⁸ with either a graphene sheet or in the presence of a Cor/TP4 sensitized layer.

This strategy is consistent with previous computational studies, which have proven the ability of molecular modeling and simulation to describe the response of solute-gated sensors.⁵⁹⁻⁶²

Unexpectedly, as reported in the SI, we note that G4 is only barely interacting with the naked graphene layer, as strikingly evidenced by the binding free energy profile also reported in the SI. This behavior is not unexpected due to the high density
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<figure><img src="image_15.png" /><figcaption>**Figure 4.** Representative snapshot showing the interaction between the G4 and the Cor/TP4 sensitizing the graphene sheet, in the surface perpendicular binding mode. Density profile of the ion distribution along the axis perpendicular to the graphene sheet for isolated graphene (B), the graphene-bound Cor/TP4 (C), and the graphene-bound Cor/TP4 interacting with the G4 (D).</figcaption></figure>

of negative charges of the G4 backbone, which disfavor its approach to the hydrophobic graphene surface. Conversely, in the case of the Cor/TP4 sensitized surface, the G4 is rapidly and persistently locked in an interaction with the TMPyP4 unit (Figure 4). In order to fully simulate the G4 recognition in a realistic medium, we decided to initially place the nucleic acid in the water bulk in a region far from both the Cor/TP4 and the graphene layer, while enforcing a 0.15 M concentration of NaCl (see the SI). The spontaneous binding of the nucleic acid is not unexpected due to the known affinity of TMPyP4 and takes place in less than 100 ns. Note, however, that the binding of G4 to Cor/TP4 does not lead to any significant structural deformation of the G4, which remains close to its ideal structure. In particular, no evidence of induced transition between different G4 topologies can be pointed out. The binding is mostly driven by electrostatic interactions between the positive ligand and the negative DNA backbone, as well as by favorable π-stacking with the tetrad plans. These results are also coherent with the well-known strong affinity of TMPyP4 for G4s.<sup>63-65</sup> As shown in Figure 4B–D, we may also evidence that the interaction of the G4 with the graphene-bound Cor/TP4 correlates with a significant alteration of the ion density profile. No significant charge accumulation can be observed in the case of a naked graphene sheet (Figure 4B) or a G4 in the presence of graphene (SI). Conversely, the graphene Cor/TP4 sensitized systems present a slight accumulation of anions close to the surface due to the interaction with the positive charges of TYMPyP4 (Figure 4C). This arrangement is, however,

completely disrupted when a G4 is interacting with the graphene bound Cor/TP4. Indeed, we may now observe a very pronounced accumulation of cations in the proximity of the graphene surface in the range between 5 and 25 Å, their ratio being almost doubled. The observed different charge distribution around the graphene layer, i.e., the semiconductor, clearly represents the proof of concept that locking of G4 will lead to an electrical response of the sensor. The strong discontinuity and the important difference in the charge distribution should also be related to a high sensitivity of the device. At this stage, we cannot provide a full mapping of the real device response, even though, given the observed difference in the ion distribution, we may hypothesize that the change in the recorded signal should be observed. Questions may still arise concerning the selectivity toward other DNA or RNA structures, such as aptamers or double helical structures. However, TMPyP4 is known to interact preferentially with G4, which should, therefore, be sensed preferentially over other arrangements. The possibility to modulate the graphene sensing moiety to tune the selectivity of the sensor may also be seen as a potential interesting feature of our model. Interestingly, as reported in the SI, we have also proven that the G4 may bind to the Cor/TP4 even when the latter is lying flat on the graphene surface, i.e., the second observed conformations reported in Figure 3. More importantly, the binding of the G4 also correlates with a strong perturbation of the cations’ density profile in the vicinity of the graphene surface. In turn, these results suggest that our
proposed SGGTs should be operative whatever the binding mode on the graphene surface. However, in the case of selective G4 binders we may anticipate that the conformational state in which the G4 ligands are perpendicular to and not interacting with the graphene sheet should be preferable to ensure a selective recognition of the nucleic acid.

In this contribution, we have presented a proof of concept for the development of original SGGT sensors for the rapid detection of G4s in different matrices. Specifically, by using MD simulations complemented with enhanced sampling techniques, we have unraveled the binding free energies of poly unsaturated aromatic moieties with a graphene sheet. Furthermore, by analyzing the free-energy profiles, we have proposed a hybrid sensitizer able to interact firmly with a graphene layer while exposing a G4-binding unit to the bulk. This leads to the possibility of anchoring the G4 near the graphene surface, which, in turn, is translated into a significant alteration of the charge density near the graphene layer, an occurrence that should induce a non-negligible response of the sensor. Our work constitutes the first proposal of SGGTs for the detection of G4 structures. Moreover, it also sketches some design rules of thumb for the possible optimization of the device. In particular, the role of the linker and its rigidity is of utmost importance to prevent the formation of unwanted stacking interactions with the graphene surface. More importantly, we provide the first evidence of the possible use of SGGTs for fast sensing of the presence of G4. Furthermore, the use of metal complexes which present selectivity toward a specific G4 structure, recently proposed in our group,⁴⁰ will be considered in follow-up work to propose the design of selective sensors.

## ASSOCIATED CONTENT

**Supporting Information**

The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jpclett.4c02796.

Extended computational details including a table summarizing all the studied systems' characteristics, pictorial definition of the collective variable, overlap of the US windows, PMF for the interaction between G4 and the naked graphene, and individual ion distribution analysis (PDF)

Structure of Cor/TP4 interacting with the graphene layer (PDB)

Structure of Cor/TP4 and the G4 interacting with the graphene layer (PDB)

Structure of anthracene interacting with the graphene layer (PDB)

Structure pyrene interacting with the graphene layer (PDB)

Structure of TMPyP4 interacting with the graphene layer (PDB)

Structure of coronene interacting with the graphene layer (PDB)

## AUTHOR INFORMATION

**Corresponding Authors**

Florent Barbault – Université Paris Cité, CNRS, ITODYS, F-75013 Paris, France; ● orcid.org/0000-0002-6082-3194; Email: florent.barbault@u-paris.fr

Antonio Monari – Université Paris Cité, CNRS, ITODYS, F-75013 Paris, France; ● orcid.org/0000-0001-9464-1463; Email: Antonio.monari@u-paris.fr

## Authors

Aurianne Rainot – Université Paris Cité, CNRS, ITODYS, F-75013 Paris, France; Università degli Studi di Palermo, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies Viale delle Scienze, 90128 Palermo, Italy; ● orcid.org/0000-0002-1860-1096

Luisa D'Anna – Università degli Studi di Palermo, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies Viale delle Scienze, 90128 Palermo, Italy; ● orcid.org/0000-0002-7046-5140

Alessio Terenzi – Università degli Studi di Palermo, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies Viale delle Scienze, 90128 Palermo, Italy; ● orcid.org/0000-0001-9751-1373

Raphael Rouget – Université de Lorraine and CNRS, UMR, F-54000 Nancy, France

Stéphanie Grandemange – Université de Lorraine and CNRS, UMR, F-54000 Nancy, France

Benoit Piro – Université Paris Cité, CNRS, ITODYS, F-75013 Paris, France

Giampaolo Barone – Università degli Studi di Palermo, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies Viale delle Scienze, 90128 Palermo, Italy; ● orcid.org/0000-0001-8773-2359

Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jpclett.4c02796

## Notes

The authors declare no competing financial interest.

## ACKNOWLEDGMENTS

This work has been performed using computational resources from GENCI national computing centers, which are gratefully acknowledged. A.R., A.M., and F.B. thank the Labex Seam of Université Paris Cité and Università degli studi di Palermo for funding her Ph.D. grant. The authors thank ANR and CGI (Commissariat à l'Investissement d'Avenir) for their financial support of this work through Labex SEAM (Science and Engineering for Advanced Materials and devices), ANR 11 LABX 086 and ANR 11 IDEX 05 02. The support of the IdEx "Université Paris 2019" ANR-18-IDEX-0001 and of the Platform P3MB is gratefully acknowledged.

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