Supramolecular Hydrogel Inspired from DNA Structures Mimics

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Supramolecular Hydrogel Inspired from DNA Structures Mimics Peroxidase Activity Tanima Bhattacharyya, Y. Pavan Kumar, and Jyotirmayee Dash ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00563 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 17, 2017

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ACS Biomaterials Science & Engineering

Supramolecular Hydrogel Inspired from DNA Structures Mimics Peroxidase Activity Tanima Bhattacharyya, Y. Pavan Kumar, Jyotirmayee Dash* Department of Organic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700032, India. Email: [email protected]

KEYWORDS: DNAzyme, enzyme mimic, guanosine, logic operation, supramolecular chemistry

ABSTRACT

We herein report that hydrogels can be prepared from guanosine and boronic acids in the presence of K+ and Pb2+. These supramolecular hydrogels are formed via G-quartet like selfassembly of guanosine and its boronate esters. The potential of this hydrogel construct in mimicking enzyme-like activity has been demonstrated for the first time. We have observed that the self-assembled structure present in K+ stabilized hydrogel binds to iron (III)-hemin and shows peroxidase activity, catalyzing oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB) in the presence of H2O2. Furthermore, the conformation of the G-quartet assemblies in the hydrogel can be altered by varying the stabilizing cations K+ and Pb2+. This conformational switching has been used to devise a molecular logic gate for sensing of toxic Pb2+ ions.

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INTRODUCTION

Bio-molecular self-assembly is an inspirational approach for the design and construction of functional soft-materials. The G-quartet (G4) formed by the interaction of four guanine bases via Hoogsteen type hydrogen bonds, is a prime example of a natural system.1-9 It is the fundamental building block of biologically relevant telomeres, promoter regions as well as the untranslated regions (UTR) of mRNAs of several proto-oncogenes.10-13 The synthetic G4 assembly provides unique possibilities to generate supramolecular architectures as well as functional materials such as gels,14-17 columnar discotic liquid crystals, cross-linked polymers and synthetic ion channels.19, 18-23

Since Bang et al24 reported that GMP can form hydrogel, several researchers have

developed methods to prepare stable hydrogels of guanine derivatives.25-40 However, their applications are not yet fully explored. Recently, Davis et al reported anion stabilized guanosine hydrogels33-36 that can be further used for the delivery of anticancer drugs. We herein report a novel G-quartet hydrogel construct, prepared from guanosine and phenylboronic acid, which mimics DNAzyme like peroxidase activity providing a three-in-one platform for catalysis, sensing and logic operation.

MATERIALS AND METHODS

Materials. Guanosine, boronic acids, lead nitrate, KOH, hemin and TMB (3,3’,5,5’tetramethylbenzidine) were purchased commercially and used without further purification. MilliQ water (pH ~ 7.4) was used in all experiments. Stock solution (of high concentration) of hemin (10 mM) and TMB (1M) were first prepared in DMSO, stored in the dark at –20 oC and required dilutions for experiments were made from the same using MilliQ water.


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Preparation of K+ stabilized boronic acid hydrogels. A mixture of guanosine 1 (10 mg, 0.035 mmol), boronic acid 2 (0.018 mmol, 0.5 equiv) and KOH (1 mg, 0.018 mmol, 0.5 equiv) in 0.5 mL MilliQ water was heated at 100 oC till a clear solution was obtained. The resulting mixture was then cooled to room temperature. Gelation occurred within 5 min to give a clear and strong reversible gel (Figure S1, S.I.) (pH ~ 12). The above condition for obtaining stable hydrogel was optimized by using different compositions of boronic acid, guanosine and KOH (Table S1, SI). Preparation of Pb2+ stabilized phenylboronic acid hydrogel. A mixture of guanosine 1 (10 mg, 0.035 mmol, 1 equiv), phenylboronic acid 2a (2 mg, 0.018 mmol, 0.5 equiv) and a water soluble salt of lead, lead nitrate (5.8 mg, 0.018 mmol, 0.5 equiv) in 0.5 mL MilliQ water was heated at 100 oC till a clear solution was obtained. The resulting mixture was then cooled at room temperature. Gelation occurred within 5 min to give a clear but weak gel that is stable for 1 h (pH: ~ 6.9). Then gradual crystallization started and the gel was completely disintegrated within 3 h. Differential scanning calorimetry (DSC). DSC was carried out with required amount of the hydrogel in the range of -10 oC to 80 oC using LVC pans in a Perkin Elmer made Diamond DSC machine at a scan rate of 2 oC min-1. Thermogravimetric analysis (TGA). TGA was performed with the hydrogel (8 mg) in a temperature range of 20-300 oC in a SDTQ600 TGA apparatus. IR spectroscopy. The FTIR spectra of the dried hydrogel, guanosine and phenylboronic acid were recorded on a PerkinElmer spectrophotometer using KBr disk techniques (Figure S5). TEM and AFM analysis. The morphology of the G-PhB hydrogel was characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM) experiments. For


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the TEM and AFM experiments, hydrogel (1.5 mg) was diluted with MilliQ water (400 µL) to obtain a transparent dispersion. TEM analysis. TEM measurements were performed on a JEM 2011 JEOL electron microscope, operated at an acceleration voltage of 120 keV. The sample was prepared by drop casting the aqueous dispersion of the hydrogel onto a carbon-coated 300 mesh copper grid, followed by drying under room temperature. AFM analysis. AFM measurements were performed using a tapping mode Veeco diCP-II AFM. Disperse solutions of the guanosine hydrogel were dropped onto freshly cleaved mica surface and dried in air at room temperature overnight. Powder X- ray diffraction (PXRD) study. PXRD experiment was carried out with a dried thin film of the gel on a glass slide using an X’Pert PRO X-ray Powder Diffractometer (PANalytical, Netherlands made) from an angle range of 0o to 100o. CD analysis of the hydrogels. CD spectra of the hydrogels (K+ and Pb2+ stabilized hydrogels) were recorded at room temperature using a 1 mm path length quartz cuvette using a JASCO J815 spectrophotometer. The CD spectra represented an average of three scans and were smoothed and zero corrected. CD analysis of the hemin incubated hydrogels. A mixture of guanosine 1 (10 mg, 0.035 mmol), phenylboronic acid 2a (2 mg, 0.018 mmol, 0.5 equiv) and KOH (1 mg, 0.018 mmol, 0.5 equiv) in 0.5 mL MilliQ water was heated at 100 oC till a clear solution was obtained. The resulting mixture was then cooled to room temperature and a solution of hemin (0.25 mM, 1 µL) in MilliQ water was added to the mixture. The Pb2+ hydrogel was similarly prepared by using guanosine 1 (10 mg, 0.035 mmol), phenylboronic acid 2a (2 mg, 0.018 mmol, 0.5 equiv) and Pb(NO3)2 (5.8 mg, 0.018 mmol, 0.5 equiv). After the formation of the Pb2+ gel (5 min), a


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solution of hemin (0.25 mM, 1 µL) in MilliQ water was added. The hemin incubated hydrogels were then allowed to stand in the dark for 30 minutes and the CD spectra were recorded. Rheological measurement. Rheological measurement of the K+-hydrogel was performed using TA-ARG2 rheometer using a steel parallel plate with 40 mm diameter at 25 °C with 1.0 mm Gap spacing. NMR spectroscopy. 1H NMR spectra were recorded at Bruker 300 MHz instrument. The NMR spectra were recorded using D2O as solvent and tBuOH as external standard. MALDI-TOF spectroscopy. MALDI-TOF mass spectrum was recorded using Bruker Daltonics flex Analyser in positive reflectron mode, using α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix. Samples (K+ hydrogel or Pb2+ hydrogel) and matrix were dissolved in water and acetonitrile at concentrations of 20 mg mL−1 respectively. Sample preparation involved depositing 1 µL of the sample and matrix mixture on the wells of a 384-well ground-steel plate and allowing the spots to dry. UV-Vis spectroscopic study of hemin incubated hydrogels. The UV spectra were recorded using a Carry 100 UV-Vis spectrophotometer, at room temperature using 1 mm path length quartz cuvette. A mixture of guanosine 1 (10 mg, 0.035 mmol), phenylboronic acid 2a (2 mg, 0.018 mmol, 0.5 equiv) and KOH (1 mg, 0.018 mmol, 0.5 equiv) in 0.5 mL MilliQ water was heated at 100 oC till a clear solution was obtained. The resulting mixture was then cooled to room temperature and a solution of hemin (0.25 mM, 1 µL) in MilliQ water was added to the mixture. The Pb2+ hydrogel was similarly prepared by using guanosine 1 (10 mg, 0.035 mmol), phenylboronic acid 2a (2 mg, 0.018 mmol, 0.5 equiv) and Pb(NO3)2 (5.8 mg, 0.018 mmol, 0.5 equiv). After the formation of the Pb2+ gel (5 min), a solution of hemin (0.25 mM, 1 µL) in MilliQ water was added. The hemin incubated hydrogels were then allowed to stand in the dark


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for 30 minutes. The UV-Vis spectra of these hemin incorporated hydrogels was recorded and compared with that of hemin. UV-Vis spectroscopic study of oxidation of TMB. The spectra were recorded in a Carry 100 UV-Vis spectrophotometer, at room temperature using 1 mm path length quartz cuvette. All spectra were zero corrected. For this study, a mixture of guanosine 1 (10 mg, 0.035 mmol), phenylboronic acid 2a (2 mg, 0.018 mmol, 0.5 equiv) and KOH (1 mg, 0.018 mmol, 0.5 equiv) in 0.5 mL MilliQ water was heated at 100 oC till a clear solution was obtained. The resulting mixture was then cooled to room temperature and a solution of hemin (0.25 mM, 1 µL) was added to the mixture. Gelation occurred within 5 min. It was then allowed to stand in the dark for 30 minutes. This hemin incorporated G-PhB hydrogel (0.065 g) was then added into a 200 µL aqueous solution containing H2O2 (176 mM, 20 µL) and TMB (0.08 M, 20 µL) solutions. Then the spectra were recorded. Kinetic experiments for the oxidation of TMB. Kinetic experiments were carried out in a Carry 100 UV-Vis spectrophotometer, at room temperature using 1 mm path length quartz cuvette. Hemin incorporated G-PhB hydrogel (0.065 g) (as described previously) was added into a 200 µL aqueous solution containing H2O2 (176 mM, 20 µL) and TMB (0.08 M, 20 µL) solutions. The absorbance of this suspension at 652 nm was recorded for a time period of 14 minutes, which is till saturation was reached. Kinetic experiment of oxidation of TMB in presence of Pb2+ ions. Kinetic experiments were carried out in a Carry 100 UV-Vis spectrophotometer, at room temperature using 1 mm path length quartz cuvette. The hemin incorporated Pb2+ gel (0.065 g) was added to an aqueous solution (200 µL) containing H2O2 (176 mM, 20 µL) and TMB (0.08M, 20 µL) solutions. The absorbance at 652 nm was recorded for a time period of 14 minutes.


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Visual detection of lead ions. For the visual detection of Pb2+ ions, 5 sets of Pb(NO3)2 solution (200 µL) of 10-3, 10-4,10-5, 10-7 and 10-9 M, were taken and the K+-hydrogel (G-PhB hydrogel) (0.065g) was added to each set. These sets were then incubated with hemin (0.25 mM, 1 µL) for half an hour. Then H2O2 solution (176 mM, 40 µL) was added to each set and allowed to stand for 15 minutes. Then TMB solution (0.08M, 40 µL) was then added to each set. UV-Vis spectroscopic study for devising a logic gate. Four experimental setups were taken for the purpose: A) Vial containing water (200 µL), B) Vial containing hemin incorporated K+ gel in water (200 µL), C) Vial containing K+ gel incubated in Pb(NO3)2 solution (1x10-4 M, 200 µL) and hemin, and D) Vial containing hemin incorporated Pb2+ gel in water (200 µL) (0.065g of gel were taken for each set). These sets were then treated with H2O2 (176 mM, 40 µL) for 15 minutes, followed by TMB solution (0.08M, 40 µL). Then the absorbance spectrum of each set was recorded on a Carry 100 UV-Vis spectrophotometer, at room temperature using a 1 mm path length quartz cuvette. All spectra were thus zero corrected. Cytotoxicity study of the K+ stabilized G-PhB hydrogel. The ability of the synthesized K+ stabilized G-PhB hydrogel to affect the viability of HeLa cancer cells was investigated using the XTT assay. HeLa cells were cultured in DMEM supplemented with 10% FBS. Cells were grown in 96-well tissue culture plate at a density of 4× 105 cells/well at 37 °C in an atmosphere of 5% CO2. After 24 h, the cells were treated with the hydrogel at different concentrations (1, 5, 10, 20, 50, 100, 200 and 500 µM, wrt guanosine content of the gel) and incubated for 24 h. 25µL of a mixture of XTT+PMS [4mL of XTT (at a concentration of 1mg/mL) and 10 µL of PMS (10 mM)] were added to each well and kept for 2 h at 37 °C/5% CO2. Absorbance (A) of formazan dye was measured at 450 nm using a microplate reader. The percentage cell viability was normalized to the absorbance of untreated cells.


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RESULTS AND DISCUSSION The G-PhB hydrogel was prepared by heating an aqueous suspension of guanosine 1 and phenylboronic acid 2a in the presence of KOH at 100 oC to obtain a clear solution that was cooled down to ambient temperature (Scheme 1, Table S1, Figure S1, Supporting Information, SI). Using conventional tube-inversion method, we observed that the mixture efficiently formed a transparent gel within 5 minutes. It is interesting to mention that the gel formation was also observed with other aryl boronic acids like 4-methoxyphenylboronic acid 2b, 4formylphenylboronic acid 2c, 4-carboxyphenylboronic acid 2d, 4-cyanophenylboronic acid 2e, 4-pyridinylboronic acid 2f, 2-naphthylboronic acid 2g and aliphatic boronic acid like isobutylboronic acid 2h (Table S2, SI). Thus, guanosine-boronic acid offers a versatile material platform for diverse applications. Further studies were carried out using the guanosinephenylboronic acid hydrogel (G-PhB).


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Scheme 1. Formation of guanosine-boronic acid hydrogels. Differential Scanning Calorimetry (DSC) showed that the gel to sol transition temperature (Tgel) of the G-PhB hydrogel is 78 oC (Figure S2, SI).41 Thermogravimetric analysis (TGA) graph showed that the major weight loss (~ 100 °C) was mainly due to water loss from the gel matrix (Figure S3, SI). It is worth noting that the gelation occurred selectively with K+ as no other monovalent cations like Li+, Na+ or Cs+ which are known to stabilize G-quadruplexes could induce a super-assembly to form a gel. As guanosine 1, soluble in hot KOH solution, precipitates out when cooled, the hydrogelation may be attributed to the formation of phenylboronate ester 3a that self-assembles to form stacked assembly of G-quartet fibers. Co-operative interaction between the boronate ester 3a and residual guanosine 1 as well as π−π stacking interactions


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within the phenyl rings in phenylboronic acid component may have further strengthened the supramolecular architecture. The FTIR analysis of the G-PhB hydrogel showed an absorption band at ∼1100 cm−1 (the stretching vibration of the B-OC bond (νB-OC) for boronate esters) suggesting the formation of boronate ester in the hydrogel network (Figure S4, SI).42 The TEM images of the hydrogel revealed a highly entangled three-dimensional network of fibers of variable width, with an average diameter of 50 nm in the gel matrix (Figure 1a, Figure S5, SI). The AFM images showed that the average height of the gel fibres is 37.5 nm (Figure 1b, Figure S6, SI). A broad peak at 26.8° [d = 0.33 nm] in the PXRD pattern of the dry hydrogel could be well related to the distance between two adjacent vertical G-quartet stacks in G-quadruplexes of DNA (Figure S7).31, 43, 44

Figure 1. (a) HRTEM image of the G-PhB hydrogel, (b) AFM image of the G-PhB hydrogel. The CD spectrum of the G-PhB gel displayed positive peaks near 295 and 275 nm and negative peaks at 284 and 270 nm, suggesting a hybrid head to head and head to tail type of stacking interactions of G-quartets in the gel network (Figure 2a).33, 45-50 An intense negative band at 304 nm with a positive band at 261 nm was also observed. The observed oppositely signed bands can


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be related to a left-handed helical stacking of G-quartets.6, 45-50 The rheological properties of the hydrogel were measured at 20 oC. The storage moduli (G’) showed a substantial elastic response when measured as a function of angular frequency (up to 102 Hz) at a fixed stress of 100 Pa, and the storage moduli (G’) values were always larger than the loss moduli (G”) values, consistent with the presence of a solid-like viscoelastic hydrogel network (Figure 2b). Oscillatory stress sweep experiments (at a frequency of 1.0 Hz) for a G-PhB hydrogel (composed of 0.035 mmol guanosine, 0.5 equivalent phenylboronic acid and 0.5 equivalent KOH in 0.5 ml water) and GBoric hydrogel33 (composed of 0.035 mmol guanosine, 0.5 equivalent boric acid and 0.5 equivalent KOH in 0.5 ml water) show lower storage moduli values for the G-Boric hydrogel compared to the G-PhB hydrogel (Figure S8). This indicates the G-PhB hydrogel is comparatively stronger than the G-Boric hydrogel.33

Figure 2. (a) CD spectra of hydrogels prepared using 0.035 mmol guanosine, and 0.5 equivalent phenylboronic acid in the presence of 0.018 mmol of the respective stabilizing cation (b) Rheology spectra of the K+-stabilized hydrogel prepared using 0.035 mmol guanosine and 0.5 equivalent phenylboronic acid in the presence of 0.018 mmol of K+ ion.


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A solution phase VT-NMR spectra of G-PhB hydrogel were recorded in D2O using t

BuOH as an external standard (Figure 3). By decreasing the temperature from 70 oC to 25 oC, an

upfield shift of the peaks was observed along with broadening of the peaks. The upfield shift in the peak positions in the NMR spectrum suggests an increase in electron density around the respective nucleus of each component as the molecules come in close proximity with progress in gelation. Another important observation is that the integral values of 1’ H of guanosine (Hb) and phenylboronate ester (Ha) were decreased with the decrease in temperature from 70 oC to 25 oC. In addition, the integral values of phenyl protons of the guanosine-phenylboronate ester were also decreased. These observations suggest the formation of a binary gel system, in which both the free guanosine (1) and the guanosine-phenyl boronate ester (3a) participate in the gel formation (Table S3, SI).27, 28 The MALDI-TOF spectrum of the hydrogel further indicated the formation of guanosine-phenylboronate ester (Figure S9, SI).

Figure 3. VT-NMR spectra of the G-PhB hydrogel


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It is worth noting that the G-quartet assembly of the G-PhB hydrogel binds to iron(III)-hemin. Both UV-Vis and CD spectra show strong interaction of the gel structure with hemin.51-53 In the UV-Vis spectra, a large hyperchromicity and red-shift of the Soret band of hemin (from 380 to 387 nm) was observed upon interaction with G-PhB hydrogel (Figure S12a, SI). These results suggest that hemin binds with the G-quartet assemblies present in the gel network using endstacking mode of binding. CD spectra showed enhancement and red shift of the band signals of the gel on interaction with hemin (Figure 2a, Figure S12b, SI). It is known that hemin binds to G-quadruplex DNA and shows catalytic peroxidase activity.51-53,58 Previously, some hydrogels59 and cyclic di-GMP60,61 had been reported to exhibit peroxidase activity on binding with hemin. We hypothesized that the structures present in the gel network in combination with hemin would be able to mimic the DNAzyme (peroxidase) activity of DNA G-quadruplex. The catalytic activity of the gel-hemin complex was then studied by monitoring the oxidation of TMB (Figure 4).52, 62-64


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Figure 4. (a) Schematic representation of the peroxidase activity of the hydrogel; (b) UV-Vis absorption spectra displaying the oxidation of TMB with time; (c) Kinetic graph of the oxidation of TMB by H2O2 under different conditions. We chose TMB over other substrates as the oxidation product of TMB is colored and stable, providing TMB with great potential in the applications of visual detection of target analytes.62-64 The gel was incubated with hemin and subsequently H2O2 and TMB were added. The colorless TMB was oxidised to a blue charge-transfer complex of the initial diamine and the diimine, oxTMB (λmax = 652 nm, Figure 4). The UV-Vis spectra showed disappearance of the


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absorption peak of TMB (λmax = 285 nm) and appearance of two peaks at 370 and 652 nm in the visible region with gradual oxidation of TMB to oxTMB. The peaks were found to reach a maximum over a period of almost 14 minutes (Figure 4b). The kinetic experiments showed a relatively slow oxidation of TMB to the blue oxTMB (Figure 4c). The gel-hemin complex is necessary for the reaction to occur as no absorbance was observed for the control setups where this complex was absent. These results show that the gel can mimic DNA quadruplex in exhibiting oxidase like activity.51-57 It is reported that Pb2+ is more efficient in stabilizing Gquadruplexes than K+ ions.54-57,62-67 We wanted to examine if guanosine and phenylboronic acid can form hydrogel in Pb(NO3)2 in place of KOH. The gel prepared with Pb2+ was weak and stable up to a maximum of 1 h (Figure S10a, SI). AFM image of the Pb2+ stabilized hydrogel showed the presence of relatively straight and long fibres of height 130 nm (Figure S10b, SI). A MALDI-TOF spectrum of this Pb2+ stabilized hydrogel (pH: ∼ 6.9) confirms the presence of GB(NO3)Ph complexes in the hydrogel matrix (Figure S11, SI). It is important to note that the gelation did not occur in the presence of other G-quadruplex stabilizing divalent cations like Ba2+, Zn2+, Mg2+ or Sr2+. The CD spectra highlight the structural differences between the G-quartet architectures stabilized by K+ and Pb2+. Consistent with the CD spectra of Pb2+ stabilized G-quadruplex structures, the long wavelength maximum for the K+ gel at 295 nm was shifted to 315 nm (by 20 nm) for the Pb2+ gel indicating Pb2+ induces anti parallel arrangement of the G-quartet units in the hydrogel (Figure 2a, Figure S14a, SI).58,65-68 The CD analysis further shows a complete disruption of the G4-assemblies upon addition of hemin to the Pb2+ gel (Figure 2a, Figure S14b, SI). Our results show that the Pb2+ gel is unable to exhibit peroxidase activity (Figure S16, S17, SI). In addition, the K+ gel, when incubated with Pb(NO3)2 failed to show peroxidase activity


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(Figure 5). This suggests that Pb2+ ions displace K+ ions in the gel network because of its higher affinity for G-quadruplexes compared to K+. These results collectively suggest that binding of hemin to the G-quartet assembly in the gel is the key factor for exhibition of DNAzyme like activity. This variation in binding properties of hemin with the gel structure on changing the stabilizing ions makes this gel system a prospective sensor for Pb2+ ions.69 The gel was then incubated with a series of solutions of varying lead ion concentrations and each is treated with hemin, H2O2, and TMB.

Figure 5. (a) Schematic representation of the switching of the supramolecular conformation of the hydrogel with variation in the stabilizing cation The sets containing lead ions in a concentration higher than 10-9 M did not show a blue coloration (oxidized TMB), which suggests that Pb2+ ions inhibited the enzymatic activity of the K+ gel at that concentration (Figure S16, SI). Thus the hydrogel-hemin system can be used for


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the detection of lead ions (Figure S16, SI). We then envisaged that a logic gate can be designed with this hydrogel on the basis of the stabilizing cation (Figure 6, Table 1). The stabilizing ions in the gel media are considered as inputs and the absorbance due to oxidised TMB at 652 nm in the UV-Vis spectra was chosen as the output. Four experimental sets were considered: A) vial containing water; B) vial containing K+ gel in water; C) vial containing K+ gel incubated in Pb(NO3)2 solution; D) vial containing Pb2+ gel in water. All these sets were incubated with equal amounts of hemin for 30 min and then treated with H2O2 followed by TMB solution. Set A, where no gel was present [input (0,0)], almost no absorbance at the desired wavelength was observed, thus giving an [output 0].

Figure 6. (a) UV-Vis absorption spectra showing a peak for absorbance at 652 nm for the set B; (b) A visual demonstration showing development of color due to oxidation of TMB occurred only for Set B where composition of the different sets were: A: vial containing water, hemin, H2O2 and TMB, B: vial containing K+ gel in water, hemin, H2O2 and TMB, C: vial containing K+ gel incubated in Pb(NO3)2 solution, hemin, H2O2 and TMB, D: vial containing Pb2+ gel in water, hemin, H2O2 and TMB; (c) Diagram for the two input INHIBIT Logic gate. The peak intensity was maximum for Set B [output 1], containing the K+ gel only [input (1,0)]. The blue coloration of Set B was also well detectable with naked eye. Set C, containing K+ gel incubated in lead nitrate solution [input (1,1)] showed no absorbance at 652 nm [output 0] as the


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Pb2+ ions from the solution must have replaced the K+ ions in the G-quartet of the gel fibers. And hence hemin could not bind to the G-quartet assemblies in the gel to promote oxidation of TMB by H2O2. As Set D contained only the Pb2+ stabilized gel [input (0,1)], the catalytic complex was not formed at all, leading to no reaction. Thus set D showed no absorbance at 652 nm [output 0]. Therefore, K+ and Pb2+ can switch between two states of aggregation of the G-quartet assemblies in the gel network ('on' and 'off′), resulting in a two-input INHIBIT logic gate behaviour.58, 70-74

INPUT 1 K+ 0

INPUT 2 Pb2+ 0

OUTPUT A652 nm 0

1

0

1

1

1

0

0

1

0

Table 1. The Truth table that represents the two-input INHIBIT logic gate that can be devised considering K+ and Pb2+ as the inputs and absorbance at 652 nm as the outputs.

Furthermore, the cytotoxicity of the gel was evaluated in HeLa cells using XTT cell viability assay. It was found that the K+ stabilized G-PhB hydrogel did not display any cytotoxicity to HeLa cells after 24 h of exposure even at a concentration 500 µM, with respect to guanosine (Figure S18, S.I.). This result indicates that the gel construct can be used for drug delivery applications.

CONCLUSION AND FUTURE PERSPECTIVES

In conclusion, we have demonstrated a bio-mimetic hydrogel construct derived from guanosine and phenylboronic acid. The supramolecular assembly present in the hydrogel can substitute for


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DNA G-quadruplexes in mimicking peroxidase enzymes. The hydrogel can detect nanomolar concentrations of lead ions. The K+ stabilized G-PhB hydrogel being non-toxic can be used for the delivery of anticancer drugs. A two input INHIBIT logic gate is constructed by switching the G-quartet assembly of the gel net work induced by two different ions. Our results describe the function of guanosine derived hydrogels as artificial enzymes79 that can provide a novel platform to construct innovative supramolecular architectures for sensing, targeting drug delivery and biomolecular computation.71-78 Furthermore, the hydrogel properties can be modulated by using boronic acids with different functional groups. Such investigations are currently underway in our laboratory.

AUTHOR INFORMATION Corresponding Author *Department of Organic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700032, India. Fax: +91-33-2473-2805; Tel: +91-33-2473-4971, ext. 1405; Email: [email protected] ACKNOWLEDGMENT We thank Department of Atomic Energy (BNRS) and DST India for research funding. Authors thank Ritapa Chaudhuri for her help during the preparation of the manuscript. JD thanks DST for a SwarnaJayanti fellowship. TB thanks CSIR-India for a research fellowship. ASSOCIATED CONTENT


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Supporting Information. Additional experiments, characterization data of gels, UV-Vis spectroscopic study, spectrophotometric analysis of the study of oxidation of TMB, and cytotoxicity data. This material is available free of charge via the Internet at http://pubs.acs.org.

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TABLE OF CONTENTS GRAPHIC Supramolecular Hydrogel Inspired from DNA Structures Mimics Peroxidase Activity Tanima Bhattacharyya, Y. Pavan Kumar, Jyotirmayee Dash*


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Supramolecular Hydrogel Inspired from DNA Structures Mimics

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