Enhanced Rigidity Changes Ultraviolet Absorption: Effect of a Merocyanine Binder on G-Quadruplex Photophysics

The urge to discover selective fluorescent binders to G-quadruplexes (G4s) for rapid diagnosis must be linked to understand the effect that those have on the DNA photophysics. Herein, we report on the electronic excited states of a bound merocyanine dye to c-Myc G4 using extensive multiscale quantum mechanics/molecular mechanics calculations. We find that the absorption spectra of c-Myc G4, both without and with the intercalated dye, are mainly composed of exciton states and mixed local/charge-transfer states. The presence of merocyanine hardly affects the energy range of the guanine absorption or the number of guanines excited. However, it triggers a substantial amount (16%) of detrimental pure charge-transfer states involving oxidized guanines. We identify the rigidity introduced by the probe in G4, reducing the overlap among guanines, as the one responsible for the changes in the exciton and charge-transfer states, ultimately leading to a redshift of the absorption maximum.

G -Quadruplexes (G4s) are noncanonical secondary structures formed in nucleic acids where groups of four guanines interact via Hoogsteen base-pairing to form square structures, tetrads, that stack and are stabilized by a central metal cation, 1,2 see Scheme 1. The formation of G4 motifs appears throughout the human genome and evinces essential functions in transcription, replication, stability, epigenetic regulation, as well as in cancer formation. 3,4 G4s are present in the promoter regions of oncogenes, like in the protooncogene c-Myc, 5 which regulates several elongation factors in cellular transcription. They also appear in viruses 6 and have been discussed in the context of the pathogenicity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) currently ravaging the world. 7 While in the presence of UV radiation DNA/RNA nucleobases are able to efficiently dissipate the gained energy into heat into the environment, 8 G4s can be damaged by the generation of guanine radical cations, 9−13 thereby potentially affecting regulation of the transcription by c-Myc. 14 Conversely, there is a strong drive to exploit the fluorescence of G4s in order to develop biomarkers for rapid non-destructive diagnosis. 15 In this sense, small-molecule fluorescent probes are highly desirable tools to develop real-time diagnostics and also to monitor photo-oxidative lesions, 6 as well as to understand the photophysics and photochemistry of G4 motifs themselves. 16,17 However, the binding of chemical probes can affect the intrinsic chemical and physical properties of the G4, in both its electronic ground and excited states. Thus, only if the nature of the perturbation (structural, chemical, physical) induced by the probe is clearly identified is it possible to understand the native properties developed in G4 after light excitation. In other words, it might be possible to connect the difference in the absorption after binding, with the perturbation induced by the fluorescent probe.
The number of available fluorescent binders with preference for quadruplex over duplex structures is very reduced 18 and studies rationalizing the effect of the G4-binding are very scarce, 19 hampering the design of G4-fluorofores with optimal properties. 16,20 Most theoretical studies on the absorption of G4 have focused on unprobed G4s so far. 21−24 There exist calculations of UV/vis spectra of telomeric G4 structures, 23,24 but the spectroscopic properties of G4−probe complexes are only used as a G4 diagnostic tool to assess whether the probe binds. 25 Molecular dynamics studies have been carried out with G4 binders 26−28 with the aim to monitor the spatiotemporal status of G4s by means of fluorescent probes and shed light into the biological role of these DNA structures. In this Letter, we follow a different approach. We study the photophysics of a G4 structure, in particular c-Myc G4, in the presence and absence of a spiropyran probe in order to quantify the effect of the binder on the nature of the electronic excited states of the G4 and to correlate the differences found with the structural effects induced by the G4 binder.
Spiropyrans belong to a class of organic photochromic molecules that, depending on UV and visible light, can reversibly convert into a ring-opened merocyanine form. Intriguingly, the recently developed spiropyran decorated with a quinolizidine moiety (QSP, Scheme 2) does not bind DNA, but once it isomerizes to its open and protonated merocyanine form (QMCH), it binds strongly with c-Myc G4 in vivo. 29 The closed QSP form emits at 458 nm and after QSP isomerizes to QMCH in the presence of c-Myc G4, its emission is drastically shifted to 610 nm. This visible change from blue to red allows the in vivo detection of G4 DNAs by the QSP/QMCH system. We recently investigated the QSP → QMCH isomerization reaction mechanism and the most probable binding mode of QMCH to G4, showing that QMCH "rigidifies" G4, reducing its conformational flexibility. 30 In this binding mode, the probe is stacked to the upper tetrad of the G4 pocket, at the 3′-end, interacting mainly with the four guanines via π−π stacking, but also with the side nucleobases with non-covalent interactions 30 (see Figure 1c). Without the probe, the guanines of G4 present high mobility, as shown in Figure 1a, but the presence of QMCH reduces the mobility of G4 so that the guanines stack more compactly and have less degree of movementnotice the unoccupied space between bases in Figure 1b, c. We hypothesize that the change on the mobility of c-Myc G4 may alter the overlap between the guanine electron densities, influencing the nature of its excited states, which is in line with recent experiments that suggest 31 that restrained conformational changes are more important than the nature of the central cation or the folding topology in governing the excitation deactivation. In order to unveil this possibility, we characterize the nature of the electronic excited states of c-Myc G4 in the presence and absence of the fluorescent merocyanine probe QMCH.
Classical and mixed quantum mechanics/molecular mechanics (QM/MM) molecular dynamics were used to sample the conformational space of a c-Myc G4-folded 22-mer singlestranded DNA chain and provided 100 initial conditions on which the lowest 60 excited states are calculated with timedependent density functional theory (TD-DFT). 32 Here, an electrostatic embedding QM/MM scheme was employed, where the MM part was represented as point charges and the QM region was calculated with the CAM-B3LYP 33 functional with a def2-SVP 34 basis set. The QM region includes the 12 guanines involved in the three tetrads, the QMCH probe (see Figure 1c), and two additional frontier nucleobases to avoid spurious effects (see the Supporting Information for further details). In summary, our ad-hoc protocol relies on extensive sampling to reproduce an experimental-like ensemble of geometries and on a quantitative analysis of the electronic excitations. The high number of excitations calculated (6000 for the whole ensemble of geometries) allows for statistical analysis of the electronic effect induced by absorption of light in the G4. Ultimately, the applicability of the same protocol to both the unprobed and probed system gives the possibility of a direct comparison of the electronic excitations of the G4 in the absence and presence of the external probe.
A simple way of visualizing electronic excitations is to consider that when light absorption promotes an excited electron (E) to an upper electronic state, an electron hole (H) Scheme 1. Schematic Representation of a G-Quadruplex (G4) with Three Tetrads (M + = K + in the present study) Scheme 2. Spiropyran (QSP)−Merocyanine (QMCH) Chemical Equilibrium, Indicating that QSP Does Not Bind G4 DNA but that QMCH Does The Journal of Physical Chemistry Letters pubs.acs.org/JPCL Letter is created at the initial location of the electron. Depending on where H and E are located, the electronic excitations can be classified (see Figure 2) as (a) monomer-like or local excitations, where both H (empty circles) and E (full circles) are located on the same nucleobase; (b) exciton states, when more than one local excitation is present in more than one nucleobase; and (c) charge-transfer states, where the H and E are on different nucleobases. 35 As implemented in the wavefunction analysis software TheoDORE, 36 we additionally employ two parameters to discriminate between these states (also in Figure 2): the charge-transfer number (CT) and the number of donor (D) and acceptor (A) units. In a monomer, or local excitation, the H and E are fully localized on the same guanine unit, and thus, there is only one donor and one acceptor (D = A = 1) with no charge-transfer component (CT = 0, Figure 2a). In an exciton, the local excitations take place on different guanines, so that the number of D and A are still the same but larger than one (D = A > 1, Figure 2b). The analysis of these descriptors in the c-Myc G4 shows that in the exciton states, the involved guanines also interact with each other. This implies that a fraction of the total density transfers from one monomer to another. Thus, CT can be anything in the range 0.1 to 0.9 (Figure 2b). In contrast, pure charge-transfer states have CT > 0.9 and the H and E are separated on different D and A units, but the number of D and A is the same; they are labelled with D = A = 1 (Figure 2c). These three scenarios usually coexist after light absorption in flexible multi-chromophoric systems, as was found by studying the fluorescent behavior of human telomeric G4 DNA. 23 Here, the H and E can be localized on a different number of D and A units (D ≠ A) and different CT (0.1 < CT < 0.9) are possible. Accordingly, we label these states as mixed local/chargetransfer states (Figure 2d). In this case, some guanines are responsible of a local excitation while others induce electron density transfer between nucleobases, leading to different number of D and A participating units. Thus, these states are a combination of local, exciton, and charge-transfer states. As we will show below, this rich mosaic of excited states with diverse charge-transfer values and diverse spatial localizations will contribute differently to the UV spectrum of c-Myc G4 with or without the fluorescent probe.
The absorption spectrum of c-Myc G4 alone (without probe), obtained from 6000 excited states calculated from an ensemble of 100 geometries, is shown in Figure 3a (black line).
It displays two peaks centered at 4.8 and 5.4 eV, respectively. We additionally deconvoluted the spectrum according to the contributions given by the monomer local excitations (blue line), exciton states (violet line), and mixed states (green line). Noteworthy, pure charge-transfer states (D = A = 1, CT > 0.9) do not exist in our ensemble of vertical excitations because a small contribution of local excitations is always present throughout all the excitations. The contribution of monomer or local excitations alone is small; it peaks at circa 5.0 eV and corresponds to local guanine π → π* excitations. 37 Thus, the relevant electronic excitations underlying both peaks are excitonic and/or mixed states excitations but with an important difference: whilst the excitonic excited states are significant at lower energies, the mixed states dominate the spectrum at high energies.
We can compare the computed absorption spectrum with a convoluted spectrum of the density of excited states ( Figure  3b), which includes all excited states regardless of their brightness. We decompose it also into local, exciton, and mixed excitations. Additionally, we discriminate exciton and mixed states according to their amount of charge-transfer contributions (CT < 0.5 and CT > 0.5). As observed in the absorption   We can therefore conclude that the two peaks observed in the absorption spectrum of c-Myc G4 correspond to excited states that differ in their excitation length and in their amount of charge transfer. The first one at 4.8 eV, less intense, is dominated by local, exciton and mixed states with low chargetransfer character. The second one at 5.4 eV is mostly composed of mixed states with strong charge-transfer character between different guanines. In the molecular orbital picture it means that a high fraction of density transfer (CT > 0.5) leads to a blue-shift in the absorption peak with respect of the local π → π* guanine absorption band. 37 That signifies that the G4 acts as a H-like molecular aggregate, shifting the maximum of the absorption to the blue, with respect to the single local guanine excitation, once the guanines are compacted in the tetrads. States with small charge-transfer character can be found in any region of the spectrum, although they are most relevant at low energies. In spite of the large amount of chargetransfer character contributing to the high energy peak at 5.4 eV, something that is known to lead to dark excited states in the DNA context, 38 in c-Myc G4 this peak is the brightest thanks to the mixed local/charge-transfer character of the excitations. Figure 3c clearly shows a different absorption of the guanine in the tetrads once the probe is bound. In particular, the H-like behavior vanishes, with a maximum of the absorption shifted to 5.0 eV, in agreement to the c-MyC G4 peak observed experimentally in the presence of QMCH. 29 In order to analyze the effect of the probe in the G4 absorption, we first focus on the electronic effect induced by QMCH. Due to its extended conjugated system, QMCH is, as other cyanine systems, highly prompted to absorb UV/vis light. Indeed, in the absorption spectrum of the complex G4:QMCH, we found two bands belonging to QMCH (Table 1 and Figures S1 and S2), i.e., the first, with a strong oscillator strength (f osc ∼1) centered at 2.8 eV, and the second, between 4.5−5.9 eV, weaker (f osc < 0.1). The first absorption band represents 11% of the total excitations in the complex and appears in a region that does not overlap with G4; the second one takes 7%. Importantly, in contrast to unprobed G4, there is now a relevant population of pure charge-transfer states (16% of the total excitations, Table 1). The location of H and E reveals that these pure charge-transfer states correspond to guanineoxidized states, as the H is fully localized on the guanines (G +. ) and E on the probe (P −. ). These states are dark ( f osc < 0.006) and lie between the QMCH and G4 absorption bands (3.1−3.9 eV, Table 1), and they exist due to oxidative nature of the positively charged merocyanine. 30,39 Although these states cannot be directly populated by absorption of light, if they are accessed non-radiatively after excitation, they would lead to an oxidative damage of the genetic code. 40 We also see that the H can be differently delocalized on nucleobases according to the electronic nature of the ligand, as shown for different merocyanine derivatives intercalated in duplex A/T. 39 Additionally, it has been extensively shown that guanine radicals, as a precursor of oxidative damage of the genetic code, can be generated by absorption of low energy UV light. 41 Since G4 is a structure with propensity to form such experimentally observed guanine photo-oxidation, this would be enhanced by the interaction with the probe. On the other hand, the possible induced damage of the genetic code could have important consequences for applications in photoinduced therapy. Therefore, we believe that despite challenges, an investigation of the dynamics of such process will be of high interest in the future.
The presence of the QMCH also leads to the appearance of few mixed guanine/probe states, where the electron density can be both transferred from the guanine to the probe (G → P) and vice versa (P → G). These states show a very small oscillator strength and represent a small percentage of the total amount of excitation (3% each). Altogether, from an electronic point of view, merocyanine does not affect particularly the energy range of the guanine absorption because the largest group of excitations (60%) is represented by excited states involving only guanines (local/exciton/mixed states within G4, Table 1), whose absorption energy range is unshifted upon binding. In the following, we shall analyze in detail this region of the QMCH:G4 electronic absorption spectrum in order to discern the effect of the probe on the character of the G → G excitations. Example excitations of the G4:QMCH complex can be found in Figure S3.
We now analyze whether the number of guanines that participate in the electronic excitations changes, in the absence or presence of the probe, and whether it leads to differences in the two spectra. To this purpose, we employ the electron delocalization length (DEL) descriptor, 38 which indicates over how many guanines an excitation is delocalized (see Supporting Information). We focus on the exciton and mixed states, as they are the relevant states in the absorption spectrum of c-Myc G4. Figure 4 displays the number of guanines involved in each of the states of G4 (Figure 4a) and of G4:QMCH (Figure 4b). Unexcitingly, the differences are negligible, meaning that QMCH does not affect the DEL distributions, neither for exciton nor for mixed states. However, much more interesting is to see that the number of excited guanines is very different in the exciton and in the mixed states. While in the former, half of the population is found in two guanines (D = A = 2), the mixed states are mostly delocalized in three guanine units (ca. 30 %) but can reach up to nine different guanines, i.e., at least three tetrads can be simultaneously involved in one excitation. Excluding the role of Energy range of absorption (E, eV), oscillator strength (f osc ), and their percentage with respect of the total number of excitation (%). P is the probe (QMCH) and G is guanine.
The Journal of Physical Chemistry Letters pubs.acs.org/JPCL Letter the DEL, we can conclude that it is the rigidity imposed by the probe (recall Figure 1a, b) that is responsible for the different absorptions at 4.5 and 5.5 eV of the two systems. Figure 3c shows the computed UV absorption spectrum of G4 in the presence of QMCH (black line). It appears in the same energy range as G4 (Figure 3a), but there is an inversion on the relative intensities of the two absorption peaks. The absolute absorption maximum (∼5.0 eV) is now the lowestenergy peak, and it is more intense than the one still centered at 5.4 eV. The low-energy region though has a higher density of states (black line, Figure 3d). In detail, we see that the number of exciton and mixed states with CT < 0.5 increases at low energies (dashed lines), while at high energy, mixed and exciton states with CT > 0.5 show comparable density of states (dotted lines) as without the probe. In the same region, the net number of exciton and mixed states with CT < 0.5 decreases (dashed lines), leading to an absolute higher number of exciton and mixed states in the low energy peak. The presence of QMCH affects mainly the oscillator strength of the mixed states at high energy, reducing their brightness, and promotes the formation of exciton and mixed states of small chargetransfer character in the low energy region. The intrinsic flexibility of the G4 promotes bright excited states at high energies with a strong charge-transfer character (mainly mixed states) and, thereby, with a high transfer of electron density among the involved fragments. Nevertheless, this situation changes upon binding of the probe. There is a reduction in the absorbance of the high energy peak due to the rigidity imposed by the dye, which stabilizes excitonic and mixed states with small charge transfer, thereby red-shifting the main absorption peak to 5.0 eV. The presence of the probe induces an external perturbation, which in turn induces a different absorption of the DNA. Knowing the nature of the perturbation, in this case the reduced mobility of the guanines, we are able to trace back the properties responsible of the UV/vis absorption of the system. The overlapping of numerous chromophores leads to the presence of mixed states, combining local, exciton, and charge-transfer states, and with a strong coupling between exciton and charge-transfer states. We showed how this overlapping is necessary for the population of mixed states with high charge transfer, responsible for the absorption at 5.4 eV of the unprobed G4 and the aggregate-like behaviour. Once the probe is bound, the absorption is shifted to the red, missing this requirement for the main absorption at those energies.
In conclusion, we have investigated for the first time the UV/vis absorption spectrum of c-Myc G4. We have characterized the absorption spectrum in terms of local, exciton, and mixed local/charge-transfer states and evaluated the impact of binding of a merocyanine binder on the photophysics of c-Myc G4. The probe does not affect either the energy range of G4 guanines absorption or the extent of the delocalization of the excited states, but its oxidative nature induces the formation of guanine oxidative states. Accordingly, the binding changes remarkably the photophysics of c-Myc G4 in the UV region. The probe induces an enhanced conformational rigidity on G4, altering the yield of exciton and mixed states absorption, ultimately leading to a global redshift in the G4 absorption maximum. These finding points out the importance of the structural flexibility in the photophysics of G4 DNA structures.
The characterization of the excitations involving the G4 binder will be helpful to functionalize fluorescent probes with optimally tuned photophysical properties. For instance, if the probe is modified so that the population of mixed/chargetransfer states is promoted, this species could evolve via nonemissive pathways and potentially react with G4. On the contrary, if functionalization promotes the population of electronic states that can relax to lower energy states, then fluorescence will be reinforced. Identifying these mixed states is thus interesting to rationalize the effect on the fluorescence of the probe. This could be an attractive avenue to explore in the future, both theoretically and experimentally. Further, our results can contribute to understand the photochemistry of related G4-binders as well as motivate the study of the temporal evolution of these excited states from both computational and experimental points of view.

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpclett.0c03070. Extended computational details, additional UV spectra, and natural transition orbitals (PDF)