Peptide-Based Nanoparticle Exhibiting

Jun 28, 2017 - Designing enzyme-mimicking active sites in artificial systems is key to achieving catalytic efficiencies rivaling those of natural enzy...
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Self-Assembled DNA/Peptide-Based Nanoparticle Exhibiting Synergistic Enzymatic Activity Qing Liu,†,‡,§ Hui Wang,†,§ Xinghua Shi,*,†,‡ Zhen-Gang Wang,*,† and Baoquan Ding*,†,‡ †

CAS Key Laboratory of Nanosystem and Hierarchial Fabrication, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, P.R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P.R. China S Supporting Information *

ABSTRACT: Designing enzyme-mimicking active sites in artificial systems is key to achieving catalytic efficiencies rivaling those of natural enzymes and can provide valuable insight in the understanding of the natural evolution of enzymes. Here, we report the design of a catalytic hemincontaining nanoparticle with self-assembled guanine-rich nucleic acid/histidine-rich peptide components that mimics the active site and peroxidative activity of hemoproteins. The chemical complementarities between the folded nucleic acid and peptide enable the spatial arrangement of essential elements in the active site and effective activation of hemin. As a result, remarkable synergistic effects of nucleic acid and peptide on the catalytic performances were observed. The turnover number of peroxide reached the order of that of natural peroxidase, and the catalytic efficiency is comparable to that of myoglobin. These results have implications in the precise design of supramolecular enzyme mimetics, particularly those with hierarchical active sites. The assemblies we describe here may also resemble an intermediate in the evolution of contemporary enzymes from the catalytic RNA of primitive cells. KEYWORDS: DNA, peptide, hemin, peroxidase mimics, self-assembly

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the nucleic-acid-based catalysts exhibit low catalytic efficiencies and limited catalytic reactions, which are likely attributable to the lack of key functional groups or proper geometrical arrangement at the active sites. SELEX may improve the catalytic properties by optimizing the screened sequences, which is usually a time-consuming and unpredictable process. Furthermore, the well-defined and sophisticated structures of enzymes illustrate that evolution and the increased complexity of life may be dominated by a hybrid catalytic system that evolved from RNA. However, the chemical forms of such a hybrid system remain unknown. A self-assembled peptide nanostructure may have served as an oasis for the prebiotic chemical evolution of RNA.17 Interestingly, the effective catalysis of some contemporary RNA-based enzymes is achieved via RNA/protein collaboration, such as in the CRISPER-Cas9 system18 or aminoacyl-tRNA synthetase,19 in which case the peptide motif is highly conserved. DNA and

nzymes play vital roles in cellular functions by catalyzing biochemical reactions. High catalytic efficiency is largely attributed to the close cooperation among reactive elements in the active sites, which have been sufficiently screened and permutated by nature. Enzyme-inspired supramolecular catalysis, which harnesses multiple weak intermolecular interactions to assemble catalytic species, has attracted considerable interest and shown great potential in medicinal and industrial biotransformation applications.1−5 However, it has been a challenge to structurally and chemically reproduce the active sites of natural enzymes in supramolecular assemblies. Nucleic acid nanotechnology has provided a toolbox for the precise design of supromolecular structures and functions.6−9 With an in vitro selection (SELEX, systematic evolution of ligands by exponential enrichment) approach, catalytic nucleic acids have been produced to possess oxidase,10 peroxidase,11,12 deoxyribozyme,13,14 or ligase activities.15 These activities of nucleic acid structures support the hypothesis of “RNA world”,16 where RNA is capable of self-replication and catalyzing key metabolic pathways in primitive cells. However, © 2017 American Chemical Society

Received: May 9, 2017 Accepted: June 28, 2017 Published: June 28, 2017 7251

DOI: 10.1021/acsnano.7b03195 ACS Nano 2017, 11, 7251−7258

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Scheme 1. Self-Assembly of Guanine-Rich Nucleic Acid, His-Rich Peptide, and the Cofactor Hemin into the PeroxidaseMimicking Nanoparticlesa

a The designed nucleic acid and peptide components exhibit structurally and chemically complementary characteristics that enable the creation of the peroxidase-mimicking hierarchical active site in the artificial enzyme.

molecule (H32) can approach the folded, guanine-rich DNAzyme-I (GGGTAGGGCGGGTTGGG; DzI) from several directions (Figures S1−S3). This preliminary approach indicated the shape complementarity between these two species. A molecular dynamics (MD) simulation was subsequently carried out for the two most stable DNA/peptide conformations (Figure S4) to identify van der Waals forces as the driving force for DzI/H32 self-assembly. In buffer conditions, the self-aggregation of H32 into spherical nanoparticles was observed (Figure S5). This behavior is attributed to the intermolecular association of β-sheets of H32 as revealed by the CD results (Figure S6). The H32/DzI assembly disturbed the conformation of the DzI G-quadruplex, without significantly altering the secondary structure of H32 or nanoparticle morphologies. The ζ-potential results (Figure S7a) indicate the effective interactions of DzI with H32, which increased the negativity of ζ-potential of H32based nanoparticles significantly. The binding mode of H32 to DzI was studied by competitive binding assay (Figure S7b). Hoechst 33258 fluorescent dyes, which were classic groove binders of DNA, were lighted up upon being bound to DzI.32,33 The addition of H32 to the DzI/Hoechst complex reduced the fluorescent intensity by 36%, illustrating that the aggregates of His-rich peptide probably competitively bound to G-quadruplex of DzI through groove binding, a common mode for DNA−protein recognition.34 The scanning transmission electron microscopy (STEM) imaging and elemental mapping results revealed the successful complexation of hemin with H32 and DzI (Figure 1A,B) by quantifying the elemental contribution of P (only found in DNA) and Fe (only found in hemin) related to the total signal. These data illustrate the uniform distribution of DzI and hemin in the nanospheres. (For more TEM images, see Figure S8.) DzI and H32 can both form coordination bonds with hemin (2.26 and 2.14 Å for G- and His-hemin iron bond lengths, respectively). To understand the organization of the hybrid selfassemblies, it is essential to identify the interactions between the hemin iron and the self-assembled DzI and H32 components. We investigated this using UV−vis spectroscopy (Figure 1C). The H32/hemin assembly exhibited a Soret band at 414 nm, and a pronounced band at 530 nm and shoulder at 570 nm. The spectral features are attributed to the imidazole → iron charge-transfer transition and the low-spin six-coordinated

RNA exhibit exquisite shape and functional group complementarity to self-assembled peptides to form DNA- or RNAbinding protein complexes.20,21 This latter fact has inspired us to design self-assembled nucleic acid/peptide hybrid systems with synergistic and tunable catalytic behaviors. Nature has evolved enzymatic active sites where the distribution of essential functional groups follows precise spatial configuration for effective activation of the catalytic center, such as in ferrochelatase,22 formate dehydrogenase,23 heme-copper oxidase,24 and hemoproteins.25 Iron(III) protoporphyrin IX (hemin) is present in hemoproteins as a cofactor. In horseradish peroxidase, a common hemoprotein, hemin as a cofactor is activated by H2O2 via the cooperation among a proximal His ligand, a distal His, and a distal Arg residue to form the primary active species, compound I.25 This species accepts the electrons from the reducing substrate, thereby leading to a variety of fundamental biological functions. The chemical groups and their spatial arrangement (Scheme S1) are prerequisites for the high catalytic efficiency. To create a similar active site facilitating the formation of compound I, here we designed a hybrid system that assembled a guanine-rich nucleic acid and a histidine (His)-rich peptide into a ligand environment for hemin, as shown in Scheme 1. The coordination, catalytic properties, and the activation mechanism of the hemin were explored. The nucleic acid strand can fold into a high-order structure composed of base-stacked quartets, which leads to hemin binding and enhancement of the peroxidase activities of hemin.26,27 The His-rich peptide provides amino acid residues for complexation with and activation of hemin.28,29 Our work reveals that the nucleic acid and peptide ligands had compositional and structural complementarities to form the enzyme-like hierarchical active site for synergistic catalytic activity, which could be easily tailored via the design of the nucleic acid and peptide molecules. Moreover, the hybrid system exhibited higher catalytic efficiency and turnover number than previously reported nucleic-acid-based peroxidase mimetics.30,31 These hybrid assemblies may even be comparable in catalysis kinetics to natural hemoproteins.

RESULTS AND DISCUSSION We first modeled a single-molecule-level rigid docking using the Autodock program to show that the 32 His-containing peptide 7252

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hemin via hydrogen bonding between His and the water ligand. These features underscore the complementary roles of the DNA and peptide components in the formation of a 3D hierarchical active site and the activation of peroxidasemimicking catalysis by hemin. H2O2-mediated catalyzed oxidization of ABTS2− (2,2′azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) was studied by monitoring the time-dependent absorbance changes of the reaction product, ABTS•+, at 415 nm (the extinction coefficient is 31 100 M−1 cm−1) (Figure S11). The initial catalytic velocity (Vi) and the maximum catalytic conversion efficiency of ABTS2− (CABTS) were used to evaluate the catalytic performance. Figure 2A shows that hemin encapsulated within the DzI/

Figure 1. (A) TEM images of the DzI/H32/hemin assembly. Scale bar: 500 nm. (B) Element-resolved STEM images of the selfassembled DzI/H32/hemin nanoparticles. Scale bar: 200 nm. [Hemin]: 100 nM. [H32]: 4 μM. [DzI]: 1 μM. (C) UV−vis spectra of free hemin (black), H32- hemin (blue), DzI (red), and DzI/H32 (green)-complexed hemin. [Hemin]: 500 nM. [H32]: 4 μM. [DzI]: 3 μM.

species35,36 with two strong-field ligands (one ligand on the sixth coordination site, presumably His residue).37,38 The DzI/ hemin assembly showed a Soret band at 403 nm and chargetransfer transitions at 626 and 504 nm with a weak absorbance at 530 nm, which are characteristic spectral features of high-spin hexa-coordinated species11,39,40 with a strong-field and weakfield ligand (water) at the axial hemin coordination positions.37,41 In the DzI/H32/hemin assembly, the Soret peak of hemin underwent a blue shift from 414 nm and leveled off at 408 nm (a red shift compared to DzI/hemin), as the DzI/ H32 ratio increased. Meanwhile, the absorbance bands of DzIcomplexed hemin iron in the visible region (450−700 nm) became more evident, which indicates the preferential coordination of hemin iron to the nucleobase ligand of DzI. (For the dependence of DzI/hemin and DzI/H32/hemin spectra on DzI concentrations, see Figure S9.) Enhanced charge-transfer transitions of hemin iron were observed, which is indicative of the stronger interactions of the dπ orbitals of the metal with the π orbitals of the pyrrole rings.42 This could be attributed to a decrease in the symmetry of the hemin moiety, which may arise from the packing forces of the peptide chains43 and the cooperative stacking effect of His residues and nucleobases on the local electric fields of hemin.44,45 Figure S10 shows the common hemin location for all of the lowest energy docking structures. The hemin stacks on the Gquartet and the exocyclic amine of the G9 base (GGGTAGGGCGGGTTGGG) lies close to and axial to the hemin iron. Within the DzI/H32/hemin assembly, the active site, in which a guanine ligand and a distal His are distributed to either side of hemin, may be rationally proposed. The neighboring His residues from the H32 aggregates can provide or accept hydrogen bonds in a role similar to the distal Arg in natural peroxidases.25,46 The heteromolecular self-assembly rigidifies the spatial location of the His residues that are catalytically important for natural peroxidase and stabilizes

Figure 2. Dependence of catalytic performance indicators Vi and CABTS of the artificial enzymes on (A) H2O2 concentration. [DzI]: 2 μM, [H32]: 4 μM. (B) DzI concentration. [H2O2]: 20 mM. [Hemin]: 100 nM. [H32]: 4 μM.

H32 assembly showed enhanced catalytic performance compared to hemin with DzI or H32 alone as the ligand, indicating a cooperation of the peptide and DNA in the catalytic activity of hemin. At high concentrations of H2O2, an inactivation of the catalytic assemblies was observed, which resulted in a stronger synergistic effect, in particular, at 20 mM H2O2. This effect is also observed in natural peroxidase subjected to excessive H2O2 and was ascribed to the formation of a reactive ferroporphyrin species followed by hemin destruction.47 The catalytic performances of the assemblies revealed a dependence on the DzI/H32 ratio (Figures 2B and S11). At 20 mM H2O2, Vi increased and leveled off at 2 μM DzI, and CABTS reached a maximum at 1.5 μM DzI. The performance of the DzI/hemin assembly was proportional to the DzI concentration within the studied range. At an appropriate DzI/H32 ratio, CABTS and Vi increased by up to 2- and 2.5-fold, respectively. Even at a DzI concentration as low as 20 nM, 5-fold lower than hemin, the assembly resulted in enhanced reaction conversion. It was suggested by Brown48 that the amino acid residues adjacent to the methylene bridges of the porphyrin ring protect peroxidase active sites against H2O2 oxidization. The end7253

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The catalytic activities of the self-assemblies are tailorable by varying the sequences of DNA or the length of the peptide ligands. We designed another four guanine-rich DNA strands adapted from DzI sequence: DzII to DzV, which can all fold into a G-quadruplex (for the sequences, see Figure S18). As shown in Figure 3A and Figure S19a, the guanine-rich DNA/

stacking of hemin at the terminals of the folded DzI results in an accessible surface at the catalytic center and causes hemin to be susceptible to H2O2,49 which accounts for the higher Vi and faster degradation rate than H32-complexed hemin. The Hisrich peptide protected hemin against oxidative degradation, a feature that is apparent in the reactions catalyzed by the H32containing assembly. The degradation of hemin at different H2O2 concentrations was also investigated by recording the time-dependent UV−vis spectral changes of hemin (Figure S13). The self-assembly of DNA with peptide decreased the H2O2-induced destruction of the DNA-complexed hemin. To investigate the enzymatic kinetics, Lineweaver−Burk plots of the initial catalytic velocity versus H2O2 concentration were constructed for the three artificial enzymes containing 100 nM hemin (Figure S14). The apparent kinetic parameters for H2O2 reduction in the presence of ABTS2− were calculated using the Michaelis−Menten equation (Table 1). The kcat

Figure 3. Effect of (A) guanine-rich DNA sequences (DzI-DzV) (1.5 nM hemin) and (B) His-rich peptide length (H9, H20, and H32) (100 nM hemin) on the catalytic performance of the DNA/ peptide/hemin assemblies. [Guanine-rich DNA]: 1.5 μM. [His-rich peptides]: 4 μM.

Table 1. Apparent Kinetic Parameters with Respect to Catalyzing Reduction of H2O2 with ABTS2− as the Reducing Substrate catalytic complexesa b

DzI/hemin H32/heminb DzI/H32/heminb DzI/H32/heminc a

kcat (s−1)

Km (mM)

kcat/Km (s−1 mM−1)

0.32 0.18 0.436 3.01

2.58 2.51 1.71 5.04

0.124 0.0717 0.254 0.597

H32/hemin assemblies exhibited activity following the order of the corresponding DNA/hemin. This is ascribed to the effectiveness of the nucleoligand coordination to hemin (Figure S19b,c). We then altered the length of the His-rich peptide component to contain 1, 9, or 20 residues (i.e., designated as H1, H9, and H20). The optimal Vi and CABTS values of the H20 complex were between those of H9 and H32 (Figure 3B; for the time-dependent courses, see Figure S20). The CD results (Figure S21) and the competitive binding assay (Figure S7b) indicate that the longer peptide interacts more strongly with DzI, which may result in a more effective synergistic activation of hemin. No synergistic effect was observed in the H1/DzI system in the catalytic activity assay or UV−vis spectra (Figure S22). The effect of the component species was also investigated using guanine-free DNA strands or His-free peptides. As shown in Figure S23, the use of noncognate DNA resulted in the inactivation of the H32-complexed hemin. Similarly, the assembly with the noncognate peptide decreased the catalytic activity of the DzI-complexed hemin (Figure S24). These results confirm the importance of the key functional groups in the nucleic acid and peptide ligands in the cooperative activation of hemin-catalyzed reactions. In addition to ABTS2−, the synergistic effects in the catalyzed oxidization of TMB (3,3′,5,5′-tetramethylbenzidine), phenol, homovanillic acid (a major catecholamine metabolite), dopamine (a catechol derivative as a neurotransmitter), and pyrogallol were also observed for DzI/H32/hemin nanoparticles (Figures S25−S29). The kinetic parameters for the reduction of H2O2 in the presence of pyrogallol are indicated in Table S1, which also shows the kinetic parameters of other reported hemin-based catalytic systems. It is found that the kcat value for hemin complexed with DzI/H32 was higher than that for hemin conjugated with cyclodextrin,51 encapsulated in supramolecular hydrogel which contained Phe and His residues.51 Particularly, at low ratio of hemin to the DzI/H32 hybrid, the kcat and kcat/Km values were higher than hemin/ graphene conjugates52 and were closer to HRP.53 This indicated the superiority of our catalytic system based on heteromolecular self-assembly. Furthermore, in addition to

[DzI]: 2 μM. [H32]: 4 μM. b100 nM hemin. c1.5 nM hemin.

value, which indicates an enzyme’s turnover number (TON), and kcat/Km, which reflects the catalytic efficiency, are both consistent with a synergistic effect of DzI and H32 ligands in creating a highly active artificial peroxidase. The superiority of the DzI/H32/hemin assembly was further verified when the ratio of hemin to the DzI/H32 components was decreased. At a hemin concentration of 10 nM (Figure S15a,b), the optimal Vi and CABTS for the DzI/H32/hemin complex were 3.5-fold and 4-fold of those for H32/hemin and 34-fold and 84-fold of those for DzI/hemin, respectively. At 1.5 nM hemin (Figure S15c,d), the optimal Vi and CABTS for the DzI/H32/hemin were 10-fold and 20-fold those of H32/ hemin, and no activity for DzI/hemin was observed. Even in the presence of 0.2 nM DzI (a far low DzI/H32 ratio), an enhancement in the substrate conversion was observed. Moreover, as shown in Table 1, the kcat value (3.01 s−1) for the assembly containing 1.5 nM hemin was almost 1 order higher than that containing 100 nM hemin (0.436 s−1) (Figure S16) and approached that of 1.5 nM natural horseradish peroxidase (approximately 18.2 s−1). The catalytic efficiency (kcat/Km) of 1.5 nM hemin-containing complex was almost identical to that of myoglobin,50 and the kcat value was almost 2 orders higher. It is noteworthy that the kcat/Km and kcat values were much higher than that of previously reported nucleic-acidbased peroxidase mimetics generated by in vitro selection or modulation.30,31 Together these data show that the His residues can promote the activation of DzI-complexed hemin. The investigation of the effect of H32 concentration on the catalytic properties of the hybrid nanoparticles, with fixed DzI/H32 ratio (1/4) and hemin concentration (1.5 nM), indicates that the catalytic activity increased and leveled off at ca. 12 μM H32 (Figure S17). 7254

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Figure 4. QM models of the self-assembled active site involved in the formation of compound I (state iv) based on DFT/B3LYP calculations. The N atoms of two Hisβ residues and the porphyrin ring are fixed in the geometry optimization process. These results show the cooperative effect of the three His and the G9 coordination group on the H2O2 activation of hemin.

addition of hemin to DzI/H32 hybrid; Figure S32). This result reveals that the preassembly of DzI with H32 enables a proper distribution of the reactive elements and full catalytic cooperation between the DNA and peptide components.

DNA, the assembly of guanine-rich RNA with H32 and hemin exhibited synergistic catalytic activity (Figure S30). Using the lowest energy mechanism of H2O2 activation of horseradish peroxidase 46 as a paradigm, a model was constructed for the artificial active site to describe the roles of the peptide and nucleic acid components in the formation of reactive intermediate compound I based on the B3LYP54,55 functional of density functional theory (DFT; Figure 4).56−59 The coordinating G9 nucleobase and three distal His groups (one Hisα and two neighboring Hisβ residues) are arranged around the hemin (state i). H2O2 (Hα−Oα−Oβ−Hβ) is held by the Hisα/Hisβ via double hydrogen bonds, N (Hisα)···Hβ−Oβ and N−H (Hisβ)···Oβ, and an Fe−Oα bond (state ii). This is followed by the abstraction of a distal proton by Hisα from H2O2 and a flip of the Fe−Oα−Hα−Oβ moiety to the Fe−Oβ− Oα−Hα complex, which is stabilized by hydrogen bonding between Hisα (H+)/Hisβ and Hβ−Oα−Oβ groups (state iii). Then, the Hβ−Oα group on the distal side of Fe is reprotonated by Hisα (H+), and the heterolytic cleavage of the Oα−Oβ bond leads to the formation of the compound I analogue (state iv), which is facilitated by a negative charge on the exocyclic amine of the G9 base and a positive charge on Hisα (H+). The bond lengths are in line with effective coordination and the hydrogen bonds. In this model, the His groups act as the acid−base catalyst and hydrogen-bond acceptors/donors, the guanine-rich DNA (or RNA) with the stacked quartets coordinates with and stabilizes the hemin iron, and the heteromolecular self-assembly enhances the G9-Fe coordination (as revealed by the spectra in Figure 1C). The electron-withdrawing effect of the polar imidazole groups60,61 adjacent to the porphyrin ring may facilitate the electron transfer from the reducing aromatic substrate to compound I, which is consistent with the higher turnover rate of ABTS2− (kcat = 0.036 s−1) for the DzI/H32/hemin assembly (0.018 s−1 for DzI/hemin and 0.016 s−1 for H32/hemin in the presence of 100 nM hemin) (for the time-dependent absorbance changes, see Figure S31). The peptide chains also provide protection for the hemin against H2O2-induced degradation. The experimental and theoretical results substantiate the attribution of the synergistic peroxidative catalysis to the formation of an enzyme-like active site. We also examined the effect of species assembly order and found an optimal catalytic performance for DzI + H32 + hemin (i.e., the

CONCLUSIONS We have described the self-assembly of nucleic acid with peptide in the construction of enzyme-mimicking catalytic nanoparticles with tailorable activities. Employing hemin as the cofactor, the self-assembled nanoparticles exhibited significantly enhanced peroxidase-mimicking activity in the oxidization of a variety of reducing substrates, compared to solely nucleic acidor peptide-based assemblies. The synergistic catalysis was attributed to the effective cooperation between the nucleic acid and peptide components which possess complementary chemical and structural characteristics. Importantly, the spatial arrangement and functions of the reactive groups in the artificial active site resemble those in natural peroxidase. The activity of the hybrid nanoparticles is highly dependent on the guanine-rich DNA that acted as a molecular scaffold for hemin coordination and stabilization, the His-rich peptide that provided activating groups and hemin that performed the rodox catalysis. This system may serve as spectrophotometric or spectrofluorimetric biosensors for (i) sensitive in vitro detection of DNA (e.g., telomeric ordered DNA62), based on that only 0.2 nM guanine-rich DNA could result in enhancement of the substrate conversion (Figure S15d); (ii) probing the activity of proteinases (e.g., matrix metallopteinases (MMPs) or caspases) that cleaved the specific-sequence peptide bridging oligomeric His residues and altered the catalytic activities. The structural and functional complexity and diversity of our catalytic system can be enhanced through appropriate design of the core components or cofactors for effective catalytic bond cleavage or formation reactions. Our work may also provide a laboratory model for a self-assembled prebiotic intermediate between the RNA-based catalytic system and contemporary enzymes. MATERIALS AND METHODS Materials. All peptides with purity level above 98% were purchased from Shanghai ZiYu Biotech Co., Ltd. All DNA oligonucleotides (purified with dual PAGE) were purchased from Invitrogen Life Technologies (Shanghai, China). RNA (with 2′Ome modification) 7255

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ACS Nano was purchased from Shanghai GenePharma Co., Ltd., and purified with HPLC. Hemin, HEPES/HEPES sodium salt, Na2HPO4/ NaH2PO4 salts, H2O2, horseradish peroxidase (HRP), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS 2−), Hoechst 33258 dye, 3,3′,5,5′-tetramethylbenzidine (TMB), phenol, o-dianisidine, homovanillic acid, pyrogallol, and DMSO were purchased from Sigma-Aldrich. Activity Assay. As-received lyophilized DNA or peptides were dissolved in deionized water to make 100 or 400 μM stock solution and stored at −20 °C. Repetitive freeze−thawing operations should be avoided. Hemin was dissolved in DMSO to make a 100 nM to 10 μM stock solution and stored at room temperature. DNA and peptide with required concentrations were mixed with hemin for 20 min. The assembly time was determined by the CD signals of DNA (the conformation was disturbed by the peptide) and UV−vis absorbance of hemin, ensuring the assembly efficiency of the complex. Freshly prepared H2O2 and the reducing substrates were added to the solution containing the assembled complexes, and the time-dependent absorbance or fluorescence change at the reactant or the product was recorded, which were used to calculate the initial catalytic velocity and the maximum conversion efficiency. Each measurement was repeated four times. Characterizations. The samples for TEM imaging were prepared by placing 5 μL of the sample solution on a glow-discharged carboncoated grid (400 meshes, Ted Pella), followed by the wicking away of the unbound sample and solution evaporation. The TEM characterization was conducted using a Hitachi H-7700 microscope operated at 80 kV. The CD spectra were measured by a Jasco J-810 spectropolarimeter. The ultraviolet−visible absorption spectra were recorded using a UV−vis spectrophotometer (UV-2450, Shimadzu). The fluorescence was measured with a spectrofluorometer (F-4500, Hitachi, Tokyo, Japan) (for Hoechst 33258 dye, λex = 346 nm). Scanning transmission electron microscopy and the elemental mapping were performed in FEI Tecnai F20 using a high-angle annular dark-field detector, coupled with an energy-dispersive X-ray spectrometer. ζ-Potential measurements were performed using a Malvern Zetasizer Nano ZS instrument. Theoretical Simulations. Multiscale computational methods (quantum-chemical calculations, molecular docking, and molecular dynamic simulation) were applied to understand the active center by exploring the structure, dynamics, and interaction of DNA DzI, peptide H32, and hemin. The DNA model was built on the basis of Bcl-2 (pdb: 2f8U)63 by retaining the conformation of G-quadruplexes and replacing the loop nucleobases. Fe(III) protoporphyrin IX (pdb: 2QSP)64 from bovine hemoglobin were used as hemin models. All the MD simulations were performed using the Gromacs 5.0 package65,66 combined with the AMBER03 force field67 and TIP4P solvent model.68 Na+ was used as counterions to neutralize the systems, and the temperature and pressure were maintained using V-rescale thermostat69 and isothermal−isobaric ensemble.70 Particle mesh Ewald71 was employed to deal with the long-range electrostatic interactions. The peptide and DNA conformations from the MD equilibration were used for molecular docking. All the molecular docking simulations were carried out with Lamarckian genetic algorithm using Autodock 4.2.6.72 A big grid box size of 126 × 126 × 126 points with a large spacing of 0.753 Å (DNA and peptide) and 0.375 Å (DNA and heme) between the grid points was implemented, and the grid box is big enough to cover the entire surface of the DNA. The ones with lowest binding energy were selected for the detailed analysis and further MD studies. The structures of the activation center model were optimized with the Gaussian 09 program, and the stationary points were confirmed to be minima by vibrational analysis. All the calculations were carried out at the B3LYP/6-31G*54,55 level of DFT.56,58,59 For the more detailed computional methods, see notes S1−S4.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b03195. Details for computational simulations, Scheme S1, Figures S1−S31 and the corresponding captions, DNA and RNA sequences (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Baoquan Ding: 0000-0003-1095-8872 Author Contributions §

Q.L. and H.W. contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors are grateful for the financial support from National Basic Research Programs of China (2016YFA0201601), National Science Foundation China (21273052, 21573051, 11422215, 11272327, and 11672079), Beijing Municipal Science & Technology Commission (No. Z161100000116036), CAS Interdisciplinary Innovation Team, National Program for Support of Top-notch Young Professionals, Youth Innovation Promotion Association CAS, and CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, REFERENCES (1) Meeuwissen, J.; Reek, J. N. H. Supramolecular Catalysis Beyond Enzyme Mimics. Nat. Chem. 2010, 2, 615−621. (2) Dong, Z. Y.; Wang, Y. G.; Yin, Y. Z.; Liu, J. Q. Supramolecular Enzyme Mimics by Self-Assembly. Curr. Opin. Colloid Interface Sci. 2011, 16, 451−458. (3) Wiester, M. J.; Ulmann, P. A.; Mirkin, C. A. Enzyme Mimics Based upon Supramolecular Coordination Chemistry. Angew. Chem., Int. Ed. 2011, 50, 114−137. (4) Wu, L. Z.; Chen, B.; Li, Z. J.; Tung, C. H. Enhancement of the Efficiency of Photocatalytic Reduction of Protons to Hydrogen via Molecular Assembly. Acc. Chem. Res. 2014, 47, 2177−2185. (5) Lin, Y. H.; Wu, L.; Huang, Y. Y.; Ren, J. S.; Qu, X. G. Positional Assembly of Hemin and Gold Nanoparticles in Graphene-Mesoporous Silica Nanohybrids for Tandem Catalysis. Chem. Sci. 2015, 6, 1272− 1276. (6) Seeman, N. C. DNA in a Material World. Nature 2003, 421, 427−431. (7) Fu, J. L.; Liu, M. H.; Liu, Y.; Yan, H. Spatially-Interactive Biomolecular Networks Organized by Nucleic Acid Nanostructures. Acc. Chem. Res. 2012, 45, 1215−1226. (8) Wilner, O. I.; Willner, I. Functionalized DNA Nanostructures. Chem. Rev. 2012, 112, 2528−2556. (9) Albinsson, B.; Hannestad, J. K.; Borjesson, K. Functionalized DNA Nanostructures for Light Harvesting and Charge Separation. Coord. Chem. Rev. 2012, 256, 2399−2413. (10) Golub, E.; Freeman, R.; Willner, I. A Hemin/G-Quadruplex Acts as an NADH Oxidase and NADH Peroxidase Mimicking DNAzyme. Angew. Chem., Int. Ed. 2011, 50, 11710−11714. (11) Travascio, P.; Li, Y. F.; Sen, D. DNA-Enhanced Peroxidase Activity of a DNA Aptamer-Hemin Complex. Chem. Biol. 1998, 5, 505−517. 7256

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