Development of a Smart Fluorescent Sensor that Specifically

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Development of a Smart Fluorescent Sensor that Specifically Recognizes the c-MYC G-Quadruplex Ming-Hao Hu, Jingwei Zhou, Wen-Hua Luo, Shuo-Bin Chen, Zhi-Shu Huang, Ruibo Wu, and Jia-Heng Tan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05298 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019

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Analytical Chemistry

Development of a Smart Fluorescent Sensor that Specifically Recognizes the c-MYC G-Quadruplex Ming-Hao Hu‡ab, Jingwei Zhou‡a, Wen-Hua Luoa, Shuo-Bin Chena, Zhi-Shu Huanga, Ruibo Wu*a and Jia-Heng Tan*a a b

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen 518060, China

ABSTRACT: The specific sensing of an exact G-quadruplex structure by small molecules has never been reported. A fluorescent sensor based on the photoinduced electron transfer (PeT) mechanism provides possibilities for such specific, one-to-one recognition, indicated by fluorescence. In this study, we have rationally developed a PeT fluorescent sensor IZFL-2 by linking triarylimidazole and fluorescein moieties. IZFL-2 is a distinctive, smart sensor whose fluorescence is tunable by its molecular conformations. We then applied IZFL-2 to sensing G-quadruplexes and found that it could exactly distinguish the wild-type c-MYC G-quadruplex from other types of G-quadruplexes, as shown by the activation of its fluorescence. To understand this behavior, we performed various experiments, including fluorescence assays, absorption assays and multiscale molecular dynamics (MD) simulations, to thoroughly investigate the optimal binding mode of IZFL-2 in the c-MYC G-quadruplex. Then, the corresponding HOMO-LUMO of IZFL-2 were analyzed, and the results demonstrated that the PeT process of IZFL-2 is suppressed only in the wild-type c-MYC G-quadruplex via specific loop interactions, which restores its fluorescence. To our knowledge, this smart molecule provides the first example and new insights for the development of sensors specific for a particular G-quadruplex structure by utilizing intramolecular PeT-controlled fluorescence switching.

Utilizing a chemical approach to develop molecules capable of the selective recognition of biomacromolecules is the main concept behind chemical biology 1. G-quadruplexes are secondary structures formed in guanine-rich sequences that comprise a planar arrangement of four guanines stabilized by Hoogsteen hydrogen bonding 2-4. Recently, a high-resolution sequencing-based method has been used to identify over 710,000 potential G-quadruplex-forming sites in the human genome 5. Moreover, such helical structures tend to accumulate in important regulatory regions, including telomeres, oncogene promoters, and the untranslated regions of mRNA 6,7. It is believed that G-quadruplex structures might play a significant regulatory role in many biological processes and have potential applications in analytical or supramolecular chemistry 8-11. The ever-increasing interest in the Gquadruplex requires the development of selective Gquadruplex-targeting sensors. Much effort has been devoted to the search for fluorescent sensors that can differentiate the Gquadruplex from duplex DNA. Therefore, many sensors with high selectivity for the G-quadruplex over duplex DNA have been developed 12-14. However, few of them are able to selectively distinguish a particular G-quadruplex structure among the multitude of potential G-quadruplex-forming sequences 15,16. To search for a selective sensor for an exact G-quadruplex structure formed by a specific G-rich sequence, we paid attention to intramolecular photoinduced electron transfer (PeT), which is a widely applicable principle for the rational design of fluorescent sensors 17-21. PeT sensors are generally constructed by attaching fluorophores to receptors that behave as electron-donor groups 22. The PeT is made possible when

the highest occupied molecular orbital (HOMO) energy level of the free receptor lies between the HOMO and lowest unoccupied molecular orbital (LUMO) energy levels of the fluorophore. Upon noninteraction, PeT occurs from the receptor to the excited fluorophore, resulting in quenched fluorescence. When the PeT sensor interacts with the target, the target disrupts the PeT process, restoring fluorescence. For a PeT sensor to be switched on, a target must lower the HOMO energy level of the receptor or increase the distance between the receptor and the fluorophore 23,24. The PeT mechanism has been applied to explain the fluorescence behaviors of several G-quadruplex probes 25-27. Upon interaction, the G-quadruplex might recognize the receptor moiety via hydrogen bonding or π-π stacking interactions, thereby lowering the HOMO energy level of the receptor and potentially resulting in fluorescence enhancement. Notably, PeT rates can also be modulated by varying the receptorfluorophore distance and its molecular conformations, making the PeT sensor highly sensitive to its environment, and such a sensor might enable the specific recognition of an exact Gquadruplex structure. Given the distinctiveness of PeT sensors, we were interested in developing PeT sensors for G-quadruplex recognition. Thus, we designed five chemosensors in silico, each of which comprises a specified G-quadruplex-targeting moiety, a fluorophore (a pH-insensitive fluorescein derivative was selected) and a linking bridge (Figure 1A). The five Gquadruplex-targeting moieties were derived from the Gquadruplex selective ligands developed by our group, including triarylimidazole 28,29, isaindigotone, berberine, cryptolepine and quinazoline derivatives 30. As demonstrated

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above, the specific recognition of an exact target requires the PeT sensor to be fluorescently sensitive to its binding pose in the biomacromolecule. Thus, we first wanted to probe the relationships between molecular conformations and PeT possibilities for the five designed sensors.

Figure 1. (A) Structures of the five designed molecules. (B) Conformational analysis of a representative compound (IZFL-2), which could be classified into five major conformers, including “Line”, “Z”, “V”, “U” and “π-stacking” styles. A similar protocol was employed for the other four compounds. (C) Relationships between the fluorophore LUMO-HOMO energy gaps and the receptor-fluorophore center distances for the five molecules. Clearly, the LUMO-HOMO energy gaps increase gradually as the molecule conformation changes from a “Line” style to a “πstacking” style and present a linear correlation only for the compound IZFL-2, which favors PeT between the receptor and fluorophore, which might quench the fluorescence.

We computationally evaluated the structural flexibility of each molecule and then classified all the possible conformations into several representative conformers. Taking IZFL-2 as an example, a single IZFL-2 molecule could generate 1291 conformations. Through RMSD-based cluster analysis, the 1291 conformations could be further classified into five groups (Figure 1B). Then, we explored the correlations between the receptor-fluorophore distances and the corresponding frontier molecule orbital (MO) energy gaps (HOMO-LUMO) of the fluorophore for those five molecules by further quantum mechanics (QM) calculations. To some extent, the HOMO-LUMO gaps should represent the PeT efficiencies in the sensor structures. As shown in Figure 1C, IZFL-2 presents the most distinguishable linear correlation features among the candidate sensors. Apparently, the LUMOHOMO energy gaps increase gradually as the molecule conformation changes from the “Line” style to the “πstacking” style, which favors the PeT process between the

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receptor and fluorophore, leading to fluorescence quenching. In addition, although a linear correlation can also be found for the compound CRFL-2, the LUMO-HOMO energy gap change is much smaller than that in IZFL-2. Moreover, in the structure of IZFL-2, the aniline-based receptor moiety has an intrinsically high HOMO energy level, rendering its quenching favorable in the absence of a binding target. Therefore, IZFL2 was selected as a “possibly smart” fluorescent sensor for further investigation. Herein, as a simplification, we classified the conformations of IZFL-2 into “Stretching” and “Stacking”. We hypothesized that if IZFL-2 binds to a specific Gquadruplex and folds to form a “Stretching” conformation, the possible conformational conversion from “Stacking” to “Stretching” should elicit an increase in fluorescence, allowing the detection of the G-quadruplex. Subsequently, IZFL-2 was prepared by coupling the receptor and fluorophore moieties by a click reaction (see experimental details in Supporting Information), and the PeT between the receptor (1c-2) and fluorophore (2d-2) was convincingly demonstrated through fluorescence investigations carried out with the two isolated moieties (Figure S1) 25. Afterwards, we analyzed the molecular conformation of IZFL-2 and investigated its fluorescence change through several experiments. We then applied IZFL-2 to sense G-quadruplex structures and found that it could specifically recognize the wild-type c-MYC G-quadruplex. To gain insight into the specific fluorescence activation upon binding the wild-type c-MYC G-quadruplex, we thoroughly investigated the binding modes of IZFL-2 with this Gquadruplex using UV-vis absorption titrations, fluorescence assays and long-time multiscale molecular dynamic simulations. Then, to provide further explanation of the activation mechanism, we analyzed the corresponding HOMO-LUMO orbitals of IZFL-2 when binding with the cMYC G-quadruplex. EXPERIMENTAL SECTION Oligonucleotides and Compounds All oligonucleotides (Table S1) were dissolved in the appropriate buffer. The oligonucleotides were confirmed to be engaged in G-quadruplex formation by circular dichroism (CD) measurements. Stock solutions of compounds (10 mM) were dissolved in DMSO and stored at -80 °C. Further dilutions of samples to working concentrations were made with the appropriate buffer immediately prior to use. UV-vis Absorption Studies UV-vis absorption studies were performed on a UV-2450 spectrophotometer (Shimadzu, Japan) using a 1 cm path length quartz cuvette. For the titration experiments, small aliquots of a stock oligonucleotide solution were added to the solution containing IZFL-2 at a fixed concentration (5 μM) in TrisHCl buffer (10 mM, pH=7.2) with 100 mM KCl. The final oligonucleotide concentration was varied from 0 to 5 μM. Fluorescence Studies Fluorescence studies were performed on an LS-55 luminescence spectrophotometer (Perkin-Elmer, USA). A quartz cuvette with a 2 mm × 10 mm path length was used for the spectra recorded. For the titration experiments, small aliquots of a stock solution of the samples were added to the solution containing the compound at fixed concentration (1 μM) in Tris-HCl buffer (10 mM, pH=7.2) with 100 mM KCl.


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Analytical Chemistry The final concentrations of the samples varied from 0 to 10 μM. The fluorescence was measured at λex = 450 nm. Circular Dichroism Studies CD studies were performed on a Chirascan circular dichroism spectrophotometer (Applied Photophysics, UK). A quartz cuvette with a 10 mm path length was used for the recording of spectra over a wavelength range of 230–330 nm with a 1 nm bandwidth, 1 nm step size and time of 0.5 s per point. The DNA samples had a concentration of 2 µM. Computational Methods The detailed methods were provided in the Supporting Information. RESULTS AND DISCUSSION Correlation between Molecular Conformation and Fluorescence of IZFL-2

Figure 2. Absorption spectra of IZFL-2 and the two isolated moieties (1c-2 and 2d-2) in ethanol (A) and the aqueous buffer (B). The red lines (1c-2+2d-2) are the arithmetical sum of the spectra of the two isolated compounds. All the compound concentrations were 20 µM. (C) Absorption spectra of IZFL-2 (20 µM) in Tris-HCl buffer at different temperatures. (D) Fluorescence spectra of IZFL-2 (10 µM) in Tris-HCl buffer at different temperatures, λex = 450 nm. This study was performed on a circular dichroism spectrophotometer.

Through computer-aided conformation studies, we demonstrated that the PeT efficiencies in IZFL-2 vary according to its molecular conformations (Figure 1B and 1C). To further clarify the correlation between the conformation and fluorescence of IZFL-2, we performed other experiments. The absorption properties of IZFL-2 and its two moieties (1c2, triarylimidazole and 2d-2, fluorescein) were studied in ethanol and in aqueous buffer 27. In ethanol, the absorption spectra of IZFL-2 fully resembled the arithmetical sum of the spectra of 1c-2 and 2d-2, indicating their weak stacking interactions in IZFL-2 (Figure 2A). However, in aqueous buffer, we observed a 10 nm bathochromic shift with respect to the sum of spectra of 1c-2 and 2d-2, providing evidence of efficient intramolecular stacking (Figure 2B), which might promote the intramolecular PeT effect and thus quench the probe fluorescence. Such different conformations of IZFL-2 in ethanol and aqueous buffer were further confirmed by the

fluorescence spectra of this probe, showing that its fluorescence was restored in ethanol and completely quenched in aqueous buffer (Figure S2). In addition, the absorption spectra of IZFL-2 were not affected by concentration changes, suggesting that the probe did not form intermolecular aggregates in the working concentration range (Figure S3). In summary, the free IZFL-2 in aqueous buffer might present a dominant intramolecular “Stacking” conformation, and this conformation might vary in different environments. Moreover, the conformation analysis of IZFL-2 at different temperatures was performed by using 1 µs classical molecular dynamic simulations, showing that at room temperature, IZFL-2 presented a stable “Stacking” conformation, while at a higher temperature, IZFL-2 converted to a dominant “Stretching” conformation (Figure S4). To prove it, a UV melting assay of IZFL-2 was performed, which indicated that the IZFL-2 molecule underwent a hypsochromic shift (approximately 6 nm) and finally resembled the arithmetical sum of the spectra of 1c-2 and 2d-2 at higher temperatures (Figure 2C), suggesting that the “Stacking” conformation of IZFL-2 in aqueous buffer would convert to a “Stretching” conformation with increasing temperature. We also investigated the IZFL-2 fluorescence at different temperatures, showing that the fluorescence was clearly enhanced with increasing temperature (Figure 2D). These results demonstrated that the conformation change would alleviate the PeT and thus lead to fluorescence enhancement. Furthermore, to strengthen the correlation between fluorescence and conformation, we turned to β-cyclodextrin. We hypothesized that β-cyclodextrin, possessing millimolar affinity for benzene and substituted aromatic systems, would bind to the aniline groups of IZFL-2 and promote the adoption of a “Stretching” conformation by IZFL-2, thus reducing the PeT rate and enhancing the fluorescence accordingly. This expectation was confirmed by a titration experiment, showing that the addition of β-cyclodextrin to IZFL-2 induced significant fluorescence (Figure S5). Besides, we then added 1-(4-methoxyphenyl)-piperazine (MPP) to the mixture of IZFL-2 and β-cyclodextrin as a competitor for β-cyclodextrin binding 23. This resulted in a 50% decrease in fluorescence (Figure S6), further supporting our hypothesis. Taken together, these results suggested that, to switch on its fluorescence, IZFL-2 might undergo a conformational change from a “Stacking” structure to a “Stretching” structure, which offered the possibility for the recognition of a particular G-quadruplex structure. Specific Sensing of the Wild-Type c-MYC G-Quadruplex by IZFL-2 As IZFL-2 was designed as a G-quadruplex chemosensor and possessed the potential to sense an exact structure, the key issue was what type of G-quadruplexes could activate the IZFL-2 fluorescence. Therefore, the fluorescence properties of IZFL-2 were explored by fluorescence assays using a large set of 33 samples, including 30 G-quadruplexes (including parallel, hybrid and antiparallel topologies, identified by CD spectra) and 3 other structures (including single-stranded and double-stranded DNA and BSA protein, see Table S1). Interestingly, as shown in Figure 3A, we observed that only the two c-MYC G-quadruplexes (pu22 and pu18, differing in the length of the flanking sequences) induced significant fluorescence enhancement compared with the other samples that triggered weak fluorescence. These results proved that



IZFL-2 had high specificity for sensing the c-MYC Gquadruplex. Given these fluorescence properties, we carried out a detailed investigation of the specific recognition of IZFL-2 for that G-quadruplex. The G-quadruplex pu18 formed by the shortest sequence (18-mer) was used for further investigations. Such a sequence might fold into a parallel G-quadruplex structure (Figure 3B, the bases in the loops are marked with red) 31. Fluorescence titrations were then performed. As shown in Figure 3C, IZFL2 alone in buffer displayed rather weak fluorescence emission. With the gradual addition of pu18, an emission peak at approximately 520 nm became significantly enhanced. We further used the non-G-quadruplex sample prepared from the same sequence as pu18 annealed in LiCl (100 mM) or CuSO4 (0.2 mM) for the fluorescence titrations (Figure 3D) and thereby proved that it was the G-quadruplex structure that induced the fluorescence (Figure 3E). We hypothesized that when free, the high electron density of the triarylimidazole moiety would promote PeT, which is known to quench fluorescein fluorescence. Conversely, the presence of the Gquadruplex pu18 might weaken the PeT rate, thereby restoring the fluorescence.

Figure 3. (A) Fluorescence intensity enhancements at 520 nm of 1 μM IZFL-2 triggered by various G-quadruplex samples and BSA protein (10 μM) in Tris-HCl buffer, λex = 450 nm. Error bars represent the standard deviations of the results from three independent experiments. Inset: the structure of IZFL-2 and the sequence of the wild-type c-MYC G-quadruplex. (B) Structure model of the wild-type c-MYC G-quadruplex pu18. The bases in the loops are marked. (C) Fluorescence spectra of 1 µM IZFL-2 with the stepwise addition of the G-quadruplex pu18 (arrow: 0–10 eq) in Tris-HCl buffer, λex = 450 nm. (D) CD spectra of pu18 sequence annealed at 100 mM KCl, 100 mM LiCl and 0.2 mM CuSO4 in Tris-HCl buffer. (E) Fluorescence intensity

enhancements of 1 µM IZFL-2 at 520 nm against the sample concentrations in Tris-HCl buffer, λex = 450 nm.

Furthermore, to mimic the cellular context, we again performed fluorescence titrations using pu18 in the presence of BSA protein. As shown in Figure S7, even when the concentration of BSA in the solution was relatively high, IZFL-2 could still sense the pu18 G-quadruplex. In addition, as the formation of the G-quadruplex in the c-MYC promoter is mainly caused by interactions with proteins (i.e., nucleolin) 32, we also prepared a pu18/nucleolin complex and then performed fluorescence titration using this sample, which indicated that although nucleolin somewhat affected the binding between IZFL-2 and pu18, IZFL-2 might still recognize the G-quadruplex with satisfactory fluorescence enhancement (Figure S7). Fluorescence Responses of IZFL-2 on the Loop-Mutated c-MYC G-Quadruplexes To further clarify the specific fluorescence activation of IZFL-2 by the c-MYC G-quadruplex, we conducted fluorescence assays for various loop-mutated c-MYC Gquadruplexes. According to a previous study, pu18 is the parallel-stranded G-quadruplex core formed in the wild-type c-MYC promoter 31. In its structure, there are three reversal loops bridging three G-tetrad layers and connecting adjacent parallel strands (see the structural model in Figure 3B): two of them are single-residue loops (A, loop 2, or T, loop 4), and the third (central) is a two-residue loop (GA, loop 3). There are also two one-residue flanking loops (A, loop 1 or G, loop 5). Based on the structure information, we mutated every loop of pu18 (single-point mutation) and ensured the formation of parallel G-quadruplexes (Figure S8). Subsequently, we compared the fluorescence of IZFL-2 binding to various loopmutated pu18 G-quadruplexes. The results are summarized in Table 1. It seemed that single-point mutation at loop 3, 4 or 5 dramatically decreased the fluorescence of IZFL-2/wild-type pu18, while mutation at loop 1 or 2 induced appreciable fluorescence. These results illustrated that the sensor showed high specificity for the wild-type pu18 G-quadruplex, reflected by the fact that even some minor mutations of pu18 dramatically quenched the fluorescence of the IZFL-2/pu18 complex. However, mutations at different positions have different effects on the IZFL-2 fluorescence, suggesting that IZFL-2 tended to bind to the 3′-terminal G-quartet of pu18, and mainly interacted with the 3rd and 4th loop. Table 1. Fluorescence intensities of IZFL-2 in the presence of loop-mutated pu18 G-quadruplexes. Name Sequence FI/F0 pu18 AGGGTGGGGAGGGTGGGG 14.2 pu18-1T TGGGTGGGGAGGGTGGGG 11.0 pu18-1G GGGGTGGGGAGGGTGGGG 2.2 pu18-1C CGGGTGGGGAGGGTGGGG 12.0 pu18-2A AGGGAGGGGAGGGTGGGG 7.5 pu18-2C AGGGCGGGGAGGGTGGGG 9.5 pu18-3aA AGGGTGGGAAGGGTGGGG 1.3 pu18-3aC AGGGTGGGCAGGGTGGGG 1.1 pu18-3aT AGGGTGGGTAGGGTGGGG 1.3 pu18-3bT AGGGTGGGGTGGGTGGGG 1.6 pu18-3bC AGGGTGGGGCGGGTGGGG 1.6 pu18-4A AGGGTGGGGAGGGAGGGG 1.7 pu18-4C AGGGTGGGGAGGGCGGGG 1.8


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Analytical Chemistry pu18-5A pu18-5C pu18-5T

AGGGTGGGGAGGGTGGGA AGGGTGGGGAGGGTGGGC AGGGTGGGGAGGGTGGGT

4.7 4.7 4.7

It is worth mentioning that, in contrast to the other two mutated sequences at loop 1, pu18-1G induced insignificant fluorescence. We found that the first G-stretch of pu18-1G contained 4 guanines. There are two possibilities for selecting three consecutive guanines out of four. Therefore, there might be different possible G-quadruplex topologies, making the structure completely different from the wild-type topology of pu18 and thus inducing weak emission, which again demonstrated the specific recognition of IZFL-2 for the wildtype pu18 G-quadruplex. In addition, in the pu18 sequence, there are two four-guanine stretches, and only the first three consecutive guanines of the two G-stretches involve the Gtetrad formation 31. Therefore, we mutated the last guanines of these two stretches into other bases, causing them to fold into different G-quadruplex topologies, and then tested their abilities to restore IZFL-2 fluorescence. As shown in Table S2, all the mutated sequences triggered weak fluorescence, illustrating the specific fluorescence activation of IZFL-2 by the wild-type pu18 G-quadruplex structure. Furthermore, we evaluated the response of the probe towards the full wild-type c-MYC sequence, pu27. The results are shown in Figure S9. This sequence actually contains five G-tracts and might fold into a mixture of G-quadruplexes using either the first four or the last four G-tracts. We anticipated that only one structure that resembles pu18 might induce the fluorescence of IZFL-2. Therefore, compared to pu18, pu27 triggered a weaker fluorescence response in IZFL-2. Interaction Studies of IZFL-2 with the c-MYC GQuadruplex The above experiments revealed that IZFL-2 specifically responded to the wild-type c-MYC G-quadruplex topology rather than its mutants. We were highly interested in how this specific recognition occurred. We first used absorption titrations to investigate the interactions between IZFL-2 and pu18 or pu18-3aT (These two G-quadruplexes had completely different fluorescence responses). As shown in Figure S10A, upon the addition of the G-quadruplex pu18, the absorption intensity of IZFL-2 gradually decreased with little redshift. The mutant c-MYC G-quadruplex pu18-3aT induced a similar absorption decrease but with a small redshift (approximately 3 nm, see Figure S10B). Considering the results from the conformation studies, which revealed that compared to the “Stretching” conformation, the “Stacking” conformation of IZFL-2 had a redshift in its absorption spectrum, we might conclude that IZFL-2 adopts different conformations in the presence of pu18 and pu18-3aT. We then fit the titration data to the Benesi–Hildebrand equation 33, which indicated that IZFL-2 bound to the two Gquadruplexes at a 1:1 binding stoichiometry (also proved by Job plot analysis, Figure S11) with similar dissociation constants, with KD values of 0.53 μM for pu18 and 0.63 μM for pu18-3aT. Further competition titrations proved that the different fluorescence enhancements triggered by the two Gquadruplexes were independent of their affinities with IZFL-2 (Figure S12). Thus, the contact mode between the probe and target might lead to a large difference in fluorescence rather than in binding affinity.

We also conducted fluorescence lifetime measurements (Figure S13). The results showed that IZFL-2 displayed one major quenched component when free in solution (τ1/f1=0.56 ns/99%), suggesting that in the absence of G-quadruplexes, triarylimidazole tended to quench the fluorescence of fluorescein through intramolecular PeT. When interacting with pu18, IZFL-2 displayed two long-lived unquenched species (τ1/f1=1.8 ns/73%, τ2/f2=3.5 ns/27%), suggesting that pu18 could inhibit the intramolecular PeT in IZFL-2. However, in the presence of pu18-3aT, the lifetime of the complex was similar to that of free IZFL-2 (τ1/f1=0.56 ns/99%). This trend in lifetime was found to be consistent with that in emission intensity. Considering that IZFL-2 exhibited similar affinities to pu18 and pu18-3aT, the difference in emission intensity and lifetime exhibited by pu18 and pu18-3aT was likely due to the distinctly different conformations of IZFL-2 in these two Gquadruplexes 26. Therefore, we hypothesized that binding to pu18 would substantially change the original conformation of IZFL-2, while binding to pu18-3aT had little impact on the IZFL-2 conformation. Binding Model of IZFL-2 in the c-MYC G-Quadruplex

Figure 4. Binding modes of IZFL-2 with wild-type pu18 (A), mutant pu18-3aT (B), mutant pu18-3aA (C), and mutant pu183aC (D), generated by QM/MM MD simulations.

Since the binding mode of IZFL-2 in the pu18 Gquadruplex was not clear experimentally, wild-type and mutated models (pu18-3aA, pu18-3aC and pu18-3aT) were constructed to computationally identify the possible binding modes of IZFL-2. As shown in Figure 4A, IZFL-2 was stretched out over the wild-type pu18 G-quadruplex in a “Stretching” conformation, whereas IZFL-2 was doubled over in a “Stacking” conformation when bound to the pu18-3aT Gquadruplex. For the wild-type pu18 complex, one of the Nmethylpiperazine groups directly interacted with the A10 base by CH-π stacking, and near-parallel π-π stacking also occurred between the A10 and G9 bases at loop 3. Thus, a three-layer parallel–shift “sandwich-like” conformation was formed and maintained in the MD simulations. The other Nmethylpiperazine group interacted with the T14 base at loop 4



by CH-π stacking. Moreover, the fluorophore stacked with the G18 base via π-π stacking and additional hydrogen bonding. These results were consistent with recent reports indicating that CH-π and hydrogen bonding interactions are important in the binding of ligands to G-quadruplexes 34,35. Regarding the pu18-3aT model, one of the Nmethylpiperazine groups was anchored to loop 3 by insertion into the interspace between two bases, destroying the parallel– shift π-π stacking to form I-shaped-like π-stacking and thus a hairpin-like binding mode. As a result, the hydrogen bond with G18 was broken, and the other N-methylpiperazine group oriented to the solvent instead of the T14 at loop 4, as observed in the wild type. Consequently, the flexible IZFL-2 molecule spontaneously folded into a “Stacking” style during the classical MD simulations by forming intramolecular parallel–shift π-π stacking between the 6-6-6 fused-ring of the fluorescein moiety and the triarylimidazole ring (Figure 4B). Furthermore, a similar IZFL-2 binding mode was detected in other pu18 mutants (pu18-3aA and pu18-3aC, Figure 4C and 4D). We assumed that these different binding modes of IZFL2 regulate the PeT process and lead to the restoration/quenching of fluorescence. This explanation was consistent with the results of the lifetime and emission studies. To shed light on the impact of flanking nucleotides on binding, sequences that include longer flanking loops (pu22 and its variant, G11T) were used in modeling studies. The results showed that IZFL-2 interacted with pu22 in a similar manner to pu18. The 5′-flanking nucleotides had no impact on the binding, while the 3′-flanking nucleotide (A22) was involved in the binding. However, loops 3 and 4 were still the most important binding sites, as in the case of pu18. In addition, G11T (or G11A or G11C) mutation caused IZFL-2 to form a “Stacking” conformation similar to that in pu18-3aT (Figure S14). Validation of the Binding Model To validate the computational reliability of predicting the binding pose, we also designed and synthesized three other molecules based on IZFL-2. As shown in Figure S15, compared to IZFL-2, M1 has a shorter linker; M2 has a longer linker; and M3 has a modified receptor moiety, that is, one of the N-methylpiperazine groups is removed. The detailed synthetic methods are presented in the Supporting Information. We then explored their binding modes in the wild-type pu18 G-quadruplex using the same computational methods. As shown in Figure S15A, one of the Nmethylpiperazine groups in M1 bound the A10 base, and the other interacted with the T14 base by CH-π stacking. In addition, the fluorophore stacked onto the G18 base. Thus, M1 exhibited a similar binding mode to IZFL-2 (“Stretching” conformation), suggesting the possible fluorescence enhancement of M1 when binding to the wild-type c-MYC Gquadruplex. For M2, due to the longer linker between the receptor and fluorophore moieties, the anchor site at G18 base was not solid enough, and the fluorophore was easily folded back to form a “Stacking” conformation (Figure S15B). Similarly, the lack of an N-methylpiperazine group in M3 would finally result in the breaking of the hydrogen bond with G18. Consequently, M3 also showed a “Stacking” conformation when binding to pu18, indicating the quenching of its fluorescence (Figure S15C). These data were then supported by fluorescence assays, which indicated that M1 was fluorescence-on and the other two sensors were

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fluorescence-off upon interaction with the wild-type c-MYC G-quadruplex. These results again demonstrated the reliability of our computational methods (Figure S15D). Mechanism of Fluorescence Activation of IZFL-2 upon Binding to the c-MYC G-Quadruplex To the best of our knowledge, the PeT rate is closely related to the structure flexibility and its MO features. Then, the corresponding PeT behaviors after IZFL-2 binding with the G-quadruplexes were illuminated by MO diagram analysis, as shown in Table S3. For the wild-type pu18 G-quadruplex complex, the “Stretching” molecular geometry structure and the unique three-layer parallel–shift “sandwich-like” π-π stacking interaction cause the local excitation of fluorophores from HOMO to LUMO, and fluorescence emission from LUMO to HOMO is detectable and reversible as the PeT process is forbidden (Figure 5A). In contrast, due to the “Stacking” conformation of IZFL-2 in the pu18-3aT Gquadruplex, HOMO/HOMO-1/HOMO-2 is mostly occupied by receptor π-electrons instead of the fluorophore, as observed in the wild type, and the intramolecular PeT process could occur from the fluorophore (LUMO, local excited state) to the receptor (HOMO) by a series of nonradiative paths to dissipate the excitation energy, resulting in fluorescence quenching (Figure 5B) similar to that in water (Figure S16). The HOMOLUMO energy diagrams of IZFL-2 in other pu18 mutants (pu18-3aA and pu18-3aC) also illustrated that PeT processes are prevalent as a result of a mutation on loop 3 (Table S3 and Figure S17). These theoretical results were consistent with the experimental fluorescence assays, suggesting that the fluorescence of IZFL-2 was reserved for the exact wild-type c-MYC G-quadruplex by the elimination of the PeT possibility. It is believed that the fluorescence restoration/quenching of IZFL-2 originates from its distinct binding modes to the wildtype and mutant pu18.

Figure 5. Excitation-related HOMO-LUMO energy diagrams of IZFL-2 with the wild-type (A) and mutant pu18 (B).

CONCLUSIONS Over 710,000 potential G-quadruplex-forming sites exist in the human genome and are widely distributed in many


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Analytical Chemistry important regulatory regions. G-quadruplex structures are believed to play significant regulatory roles in many biological processes. Many chemosensors with high selectivity for the Gquadruplex over duplex DNA have been developed. The current challenge is to develop new sensors with specificity for a particular G-quadruplex structure among the multitude of potential G-quadruplex-forming sequences. In this study, we virtually designed five PeT probes through linking a fluorescein fluorophore and five G-quadruplex selective ligands. Computer-aided molecular conformation analysis was first used to reveal the relationships between the PeT possibilities and molecular conformations for the five sensors and identified IZFL-2 as a distinctive, smart sensor with fluorescence that could be tuned by its molecular conformations. Fluorescence assays demonstrated that IZFL-2 could specifically recognize the wild-type c-MYC Gquadruplex in a one-to-one manner. To investigate this phenomenon, detailed binding mode studies were performed and indicated that IZFL-2 might interact with loops 3 and 4 of the pu18 G-quadruplex and then stretch to form a “Stretching” conformation. Further calculation experiments revealed that the unique binding mode to the wild-type c-MYC Gquadruplex arrested PeT in IZFL-2, ultimately leading to specific fluorescence enhancement. To some extent, this study reveals that the exploitation of the different loops that are present in G-quadruplex structures could confer selectivity for targeting sensors. In summary, we have rationally developed a chemosensor IZFL-2 that is highly specific for the wild-type c-MYC G-quadruplex by utilizing PeT-controlled fluorescence switching. Although no NMR study of the IZFL-2/pu18 complex is available because of the poor solubility of IZFL-2, and the in situ imaging of the wild-type c-MYC G-quadruplex also failed due to the single copy of the c-MYC gene, this study provides a striking example of how to design a specific fluorescent sensor for a particular G-quadruplex structure by exploiting the PeT mechanism. Besides, we believed that through modifying the G-quadruplex-targeting moiety or the fluorophore, we might acquire a PeT fluorescent sensor with better sensing selectivity and sensitivity for a particular Gquadruplex.

AUTHOR INFORMATION Corresponding Author * Ruibo Wu, email: [email protected]; Jia-Heng Tan, tel: +86-20-39943053, email: [email protected].

Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / ‡These authors contributed equally.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21672268 and 21773313), the Natural Science Foundation of Guangdong Province (2015A030306004 and 2016A030306038), the Outstanding Youth Programme of Special Support Plan in Guangdong Province (2015TQ01R342), and the Guangdong Provincial Key Laboratory of Construction Foundation (2017B030314030). Conflict of interest statement. None declared.

ASSOCIATED CONTENT Supporting Information

Additional information as noted in the text are supplied as Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Computational methods, synthetic methods of the compounds, nucleic acid sequences used in the study, additional experimental figures and tables, and NMR spectra of the compounds (PDF).

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Development of a Smart Fluorescent Sensor that Specifically

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