Allosteric control of peroxidase-mimicking DNAzyme activity with

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Allosteric control of peroxidase-mimicking DNAzyme activity with cationic copolymers Hiroki Sato, Naohiko Shimada, Tsukuru Masuda, and Atsushi Maruyama Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00201 • Publication Date (Web): 09 Apr 2018 Downloaded from http://pubs.acs.org on April 9, 2018

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Allosteric control of peroxidase-mimicking DNAzyme activity with cationic copolymers Hiroki Sato, Naohiko Shimada, Tsukuru Masuda, and Atsushi Maruyama* School of Life Science and Technology, Tokyo Institute of Technology, 4259 B-57 Nagatsuta Midori-ku, Yokohama, Kanagawa, Japan 226-8501 ABSTRACT Control of protein conformation and function, induced by the binding of an effector, plays significant roles in modulating biochemical reaction. Although the DNAzymes catalytic activity is similar to protein-based enzymes, reports of allosterically controlled DNAzymes are still limited except for aptamer-DNAzymes hybrrids.

Here, we report allosteric control of

peroxidase-mimicking DNAzyme activity using cationic copolymers. The DNAzyme requires a structured G-quadruplex core and hemin for activity, and the DNAzyme with a parallel Gquadruplex core has higher DNAzyme activity than DNAzymes based on other types of structure. We previously reported that a cationic copolymer composed of a cationic backbone and hydrophilic dextran side chains selectively stabilizes parallel G-quadruplex structures. In this study, we investigated effects of the cationic copolymer on peroxidase-mimicking DNAzyme activity. The cationic copolymer enhanced the DNAzyme activity by more than 30-fold by stabilizing the parallel G-quadruplex structure. Furthermore, reversible allosteric control of DNAzyme activity was achieved by adding cationic and anionic polymers.

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Keywords: Cationic copolymer, Allosteric control, DNAzyme, G-quadruplex, Interpolyelectrolyte complex

1. INTRODUCTION Allosteric control is exerted when effector molecules bind to a region other than the active site of an enzyme to alter function. The effector molecule induces a conformational change between active and inactive states. Many protein-based enzymes, including enzymes involved in metabolism and signal transduction, are allosterically controlled.1 Certain DNAs, known as DNAzymes, can catalyze specific chemical reactions similarly to protein enzymes.2,3 Compared to protein enzymes, DNAzymes have advantages such as chemical and thermal stability and ease of synthesis and modification.

DNAzymes have been investigated as

biosensors, catalysts, and DNA nanomachines.4–6 Allosteric control of DNAzyme is effective to improve the performance of many biosensors and catalysts. To date, allosteric DNAzyme systems such as aptamer-DNAzyme hybrids have been reported.7–9 Peroxidase-mimicking DNAzymes, which have a G-quadruplex-based structure and require hemin, catalyze oxidation of substrates with H2O2.3,10

The DNAzyme activity is

dependent on the G-quadruplex structures.11 Intramolecular parallel G-quadruplex structures have higher DNAzyme activity than those with intramolecular antiparallel or intermolecular parallel G-quadruplex structures because of the accessibility of hemin to G-quadruplex. We focused on the relationship between the DNA G-quadruplex structures and DNAzyme activity to allosterically regulate the enzymatic activity using a cationic copolymer, which has a DNA chaperone-like activity, as an allosteric inducer.


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We have been interested in inter-polyelectrolyte complexes (IPECs) between anionic biopolymers and cationic copolymers composed of a cationic main chain and hydrophilic graft chain such as poly(L-lysine)-graft-dextran (PLL-g-Dex) (Figure 1).12 Cationic copolymers form fully soluble IPECs with negatively charged DNA, promoting DNA duplex, triplex, and quadruplex formation because the cationic copolymers shields electrostatic repulsion of DNADNA strands.12–15 The copolymer works as a DNA chaperone that induces proper assembly of stable DNA structures from heterogeneous kinetically trapped mixtures of structures.16 We also CH

CH

CH

CH

2 2 n parallel m reported that cationic copolymers selectively stabilized the G-quadruplex structure of

CH 2

+

17

human telomeric DNA in K buffer. O

CH 2

+ Furthermore,NH reversible transformation NH 3+Cl- of stem-loop and 2

OH

OH transition were OH dimer structures or B-A achieved by alternatively adding cationic and anionic OH

O OH l OH OHIPECs with cationic copolymers within few polymers because added anionic polymers form

seconds, leading to dissociation of PLL-g-Dex from DNA.18,19

H N

H C

O

O C

H C

O C

CH 2

CH 2

CH 2

CH 2

CH 2

CH 2

CH 2

CH 2

NH 3+

NH 2+

OH OH

n

H N

OH

m

OH OH

O

l

OH

OH

Figure 1. Chemical structure of the cationic copolymer PLL-g-Dex.


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On the basis of these previous studies, we hypothesized that cationic copolymers would allosterically regulate the activity of the peroxidase-mimicking DNAzyme by stabilizing the parallel G-quadruplex structure of the DNAzyme. In this report, we evaluated the effects of PLL-g-Dex on the structures adopted by various G-quadruplex-forming DNA sequences and demonstrated that PLL-g-Dex acts as a positive effector of the DNAzymes. Furthermore, we demonstrated reversible allosteric control of DNAzyme activity by using PLL-g-Dex and an anionic polymer, which acted as a neutralizer of cationic polymers.

2. EXPERIMENTAL SECTION 2.1 Materials Poly(L-lysine hydrobromide) (PLL-HBr, Mw=7.5×103) and poly(vinylsulfonic acid, sodium salt) (PVS) were provided from Sigma (St. Louis, MO, USA).

Dextran (Dex,

Mw=8.0×103-1.2 ×104) was purchased from Funakoshi Co. (Tokyo, Japan). Hemin, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and L-tryptophan were purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan).

Dimethyl sulfoxide (DMSO), TritonX-100 and

hydrogen peroxide (H2O2) aqueous solution (30 wt%) were purchased from Wako Pure Chemical Industries (Osaka, Japan). A hemin stock solution (2 mM) was prepared in DMSO and stored in the dark at -20oC. Other solutions were prepared with deionized water purified by a Milli-Q system (Millipore, USA).

PLL-g-Dex was synthesized according to a previous report as

follows.20 Dextran was conjugated to amino groups of PLL by reductive amination reaction. The copolymer was purified by ion exchange followed by dialysis against water, and then lyophilized. The copolymer was characterized by 1H NMR measurements. Dextran content of


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PLL-g-Dex used in this study was 90 wt%. The average numbers of lysine repeating units and dextran grafts of the copolymer were 62 and 8.7, respectively. All DNA strands were purchased from Fasmac Co., Ltd. (Kanagawa, Japan) and used without further purification.

2.2 Catalytic kinetics experiments The initial velocities (Vobs) of peroxidase-mimicking DNAzyme reaction in the absence or presence of the cationic copolymer (PLL-g-Dex) were monitored in phosphate buffer (25 mM NaH2PO4/Na2HPO4, 100 mM KCl or NaCl, 0.015% Triton X-100, 1% DMSO, pH 7.0) containing 0.5 µM hemin, 0.5 - 1.5 µM DNA, 100 µM ABTS or 10 µM L-tryptophan, and 200 µM H2O2. Unless otherwise noted, the ratio of concentrations of positively charged amino groups of PLL-g-Dex to negatively charged phosphate groups of DNA (N/P ratio) was fixed at 10 because PLL-g-Dex concentration dependency of the peroxidase-mimicking DNAzyme activity is saturated at N/P of 10 (Figure S1 in Supporting Information). Reactions were initiated by the addition of H2O2. The increase of absorbance at 414 nm was measured by V-630 spectrophotometer (Jasco, Tokyo, Japan). The Vobs values were calculated from the slope of the initial linear portion of the absorbance increase. The Δε value of ABTS used was 36000 M-1 cm13

.

Based on Michaelis-Menten equation, kinetic parameters (Km, Vmax) were obtained by

calculating the Vobs against different concentrations of H2O2 (0 - 5 mM). The kcat values were obtained from the equation kcat = Vmax/[DNA].

2.3 Circular dichroism (CD) spectra


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DNA (3.0 µM) alone or DNA (3.0 µM) with cationic copolymers in phosphate buffer (25 mM NaH2PO4/Na2HPO4, 100 mM KCl or NaCl, pH 7.0) were analyzed by CD (220 - 320 nm) on a J-820 CD spectrometer (Jasco) using 10-mm quartz cells and a scan rate of 100 nm/min with a response time of 0.5 s. Data were averaged over 16 accumulations for each experiment. The CD spectra for two separate samples were measured, and averaged. CD melting curves were obtained at 260 nm or 290 nm on J-820 CD spectrometer at heating rate of 1.0 °C/min. Data were collected over the temperature range from 20 to 90 °C and fitted with Spectra Manager (Jasco).

2.4 Measurement of dissociation constant (Kd) of hemin to PS2.M The dissociation constant (Kd) of hemin from PS2.M in the absence or presence of PLLg-Dex in phosphate buffer (25 mM NaH2PO4/Na2HPO4, 100 mM KCl or NaCl, 0.015% Triton X-100, 1% DMSO, pH 7.0) was investigated as described previously with a few modifications.21 Hemin (1.0 µM) and PS2.M (0-3.0 µM) were incubated for 30 min at room temperature, and the UV-vis spectra were obtained using V-630 spectrophotometer. The Kd values of PS2.M-hemin complexes were obtained by plotting the absorbance of hemin at 404 nm against the concentrations of PS2.M. The Kd value was determined using the following equation: [DNA]0 = Kd (A-A0)/(A∞-A) + [hemin]0 (A-A0)/(A∞-A0) where [DNA]0 is the concentration of PS2.M; [hemin]0 is the concentration of hemin; A∞ and A0 are the absorbance of hemin at 404 nm in the presence of a saturating concentration of PS2.M


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and in the absence of PS2.M, respectively. A is the absorbance of hemin at 404 nm in solution with a certain concentration of DNA.

2.5. Spectrophotometric determination of pKa The pKa values for the PS2.M/hemin complexes without or with of PLL-g-Dex were measured according to a previous report.22 Hemin (2.0 µM) and PS2.M (2.0 µM) in phosphate buffer (10 mM NaH2PO4/Na2HPO4, 100 mM KCl or NaCl, 0.015% Triton X-100, 1% DMSO) without or with of PLL-g-Dex were incubated for 30 min at room temperature. The UV-vis spectra were measured using V-630 spectrophotometer in the pH range 4-11.5. pH of the solution was controlled by titration with 1 M NaOH or HCl.

3. RESULTS AND DISCUSSIONS 3.1 Effect of PLL-g-Dex on the activity of the DNAzymes We firstly investigated the effects of PLL-g-Dex on DNAzyme activity using PS2.M, which is one of the most widely investigated DNAzymes (Table 1).3,23,24 The peroxidasemimicking DNAzyme activity in the absence or presence of PLL-g-Dex was evaluated by oxidation of ABTS with H2O2. When ABTS is oxidized, absorbance at 414 nm increases. As shown Figure 2(a), absorbance at 414 nm slightly increased over time in the absence of PLL-gDex, and the absorbance quickly increased in the presence of PLL-g-Dex. PS2.M with PLL homopolymer showed lower DNAzyme activity than PS2.M alone. Note that PLL-g-Dex alone or hemin alone had no catalytic activity (Figure S2 in Supporting Information). These results indicated that PLL-g-Dex, which has abundant hydrophilic graft chains, effectively enhanced the


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DNAzyme activity in phosphate buffer containing 100 mM KCl. The PLL homopolymer likely caused aggregation of the anionic PS2.M, resulting in the decrease in the enzyme activity. We further investigated effect of PLL-g-Dex on the PS2.M/hemin DNAzyme activity using tryptophan as an ampholytic substrate.10 It was found that oxidization of tryptophan by the DNAzyme was also augmented by PLL-g-Dex (Figure S3 in Supporting Information). Thus, the enhancement of the DNAzyme activity by the cationic comb-type copolymer is not specific for anionic ABTS substrate, but would be general for other substrates.

Table 1. The DNA sequences and structures of the G-quadruplex cores of the DNAzymes used in this study. Code

Sequence

Structure

PS2.M

GTG3TAG3CG3T2G2

Coexisting or mixed type hybrid

HT

AG3(TTAG3)3

Intramolecular antiparallel

G8

T4G8T4

Intermolecular parallel

As the G-quadruplex structure is necessary for DNAzyme activity,11 we used CD to investigate the structures adopted by PS2.M in the absence and presence of PLL-g-Dex. The CD spectrum of PLL-g-Dex has no significant signal from 250 nm to 320 nm (the spectrum shown as Figure S4 in Supporting Information). CD spectra depend on the type of G-quadruplex structure. The parallel G-quadruplex has a positive peak at 260 nm and a negative peak at 240 nm, whereas an antiparallel G-quadruplex has a positive peak at 290 nm and a negative peak at 260 nm.25 In the absence of PLL-g-Dex in 100 mM KCl, the CD spectrum of PS2.M was characteristic of a mixture of parallel and antiparallel structures (Figure 2(b)). In the presence of PLL-g-Dex, the


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spectrum of PS2.M was indicative a parallel G-quadruplex structure.

Thus, PLL-g-Dex

increased the parallel G-quadruplex content in the PS2.M sample, resulting in the enhancement of the enzyme activity.

Figure 2. (a) DNAzyme reactions at 25 °C in 25 mM NaH2PO4/Na2HPO4 (pH 7.0), 100 mM KCl, 0.5 µM PS2.M, 0.5 µM hemin, 100 µM ABTS, 200 µM H2O2 with PLL-g-Dex (solid line) and without PLL-g-Dex (dotted line). (b) CD spectra of PS2.M (3.0 µM) in 25 mM NaH2PO4/Na2HPO4 (pH 7.0), 100 mM KCl with PLL-g-Dex (solid line) and without PLL-g-Dex (dotted line).

Metal cations are important for the activity of peroxidase-mimicking DNAzymes, and the DNAzyme activity is very low in the absence of K+ ions.26 In accordance with the previous study, PS2.M alone showed very little activity in K+-free 100 mM NaCl buffer. Interestingly, in the presence of PLL-g-Dex, PS2.M was active in the Na+ buffer (Figure 3(a)). The enhancement of PS2.M activity by PLL-g-Dex in the Na+ buffer is explained by stabilization of the parallel-


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stranded G-quadruplex necessary for PS2.M activity: The CD spectrum of PS2.M in the Na+ buffer without PLL-g-Dex showed a positive 290 nm peak and negative 260 nm peak, indicating that PS2.M formed antiparallel G-quadruplex structures (Figure 3(b)). The CD spectrum of PS2.M in the Na+ buffer with PLL-g-Dex had characteristic of parallel G-quadruplex. These results suggest that PLL-g-Dex promotes the formation of parallel G-quadruplex, and therefore DNAzyme activity, even in the absence of K+ ions. These experiments demonstrated that PLLg-Dex acts as a positive allosteric effector of PS2.M catalytic activity.

Figure 3. (a) DNAzyme reactions of PS2.M at 25 ○C in 25 mM NaH2PO4/Na2HPO4 (pH 7.0), 100 mM NaCl, 0.5 µM PS2.M, 0.5 µM hemin, 100 µM ABTS, 200 µM H2O2, with PLL-g-Dex (solid line) and without PLL-g-Dex (dotted line). (b) CD specta of PS2.M (3.0 µM) in 25 mM NaH2PO4/Na2HPO4 (pH 7.0), 100 mM NaCl, with PLL-g-Dex (solid line) and without PLL-gDex (dotted line).


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Figure 4. The Vobs of peroxidase-mimicking DNAzymes with different DNA sequences in 25 mM NaH2PO4/Na2HPO4 (pH 7.0), 100 mM KCl or 100 mM NaCl, 0.5 µM PS2.M, 0.5 µM hemin, 100 µM ABTS, 200 µM H2O2, without or with PLL-g-Dex.

To determine the universality of the positive effect of PLL-g-Dex on peroxidasemimicking DNAzymes having G-quadruplex structures, we assessed the DNAzyme activity with different DNA sequences in either 100 mM KCl or 100 mM NaCl without or with PLL-g-Dex (Table 1). PLL-g-Dex increased the DNAzyme ativities of HT and G8 in K+ buffer (Figure 4), in which the CD signature associated with a parallel quadruplex was increased in the presence of PLL-g-Dex (Figure S5 (a) and (b)). In Na+ buffer no significant change in DNAzyme activities of HT regardless of presence of PLL-g-Dex was observed. HT substancially adapted antiparallel quadruplex structures (Figure S5 (c)) and this structure was not changed even in the presence of PLL-g-Dex (Figure 4). These results suggested that the activity enhancement by PLL-g-Dex depends on the extent of parallel G-quadruplex stabilization.

G8 was known to adapt


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intermolecular parallel structures but its catalytic activity in Na+ buffer was considerably weak (Figure 4). Positive CD signal around 260 nm was indicative of the parallel folding of G8 in Na+ buffer (Figure S5 (d)). However, its CD peak was weak and red-shifted compared with those of G8 in K+ buffer or other sequences with intramolecular parallel quadruplex folding. Of interest, in the presence of PLL-g-Dex, the CD signal was enhanced and shifted to the longer wavelength, resulted in similar CD signals to those observed for other parallel quadruplex. Hence, PLL-gDex activated the G8 DNAzyme in Na+ buffer by rearranging its parallel quadruplex structure to a more active form. Although this rearranging effect of PLL-g-Dex was unclear, PLL-g-Dex also acted as an allosteric effector for G8 DNAzyme.

3.2 Mechanism of the enhancement of PS2.M activity by PLL-g-Dex The kinetic parameters (kcat and Km) of the DNAzyme catalyzed reactions in the absence or presence of PLL-g-Dex were estimated by measuring the kinetics in various concentrations of H2O2 (Table 2, Figure S6 in Supporting Information). The Km values of these DNAzymes were approximately 0.7–1.5 mM regardless of the presence of PLL-g-Dex. The similar Km values of all DNAzymes in this study indicated that PLL-g-Dex did not promote H2O2 binding to hemin to enhance the catalytic activity. In 100 mM KCl, the kcat value of PS2.M in the presence of PLLg-Dex was approximately 3 times larger than that in the absence of PLL-g-Dex. In 100 mM NaCl, a 35-fold peroxidase activity enhancement of PS2.M with PLL-g-Dex was observed. These kcat values of PS2.M with PLL-g-Dex are higher than those reported previously including the attempts to enhance the catalytic activity by DNA modifications such as 2’-o-methyl and by the presence of a cationic peptide.27,28


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Table 2. Effects of PLL-g-Dex on kinetic parameters of the oxidation of ABTS catalyzed by PS2.M/hemin DNAzymes, Tm of PS2.M, Kd of hemin binding, and pKa of PS2.M/hemin.

100 mM KCl 100 mM NaCl

PLL-gDex

Km (mM)

kcat (1/sec)

Tm (°C)

Kd (nM)

pKa

-

0.67

0.52

52

13

8.8

+

0.61

1.48

> 80

41

7.9

-

1.51

0.034

49 a)

630

6.8

+

0.51

1.12

62

203

7.8

a) Tm of antiparallel quadruplex was indicated.

We next investigated the relationship between kinetic parameters and the characteristics of PS2.M/hemin in the presence or absence of PLL-g-Dex. The mid-point (Tm) of the melting transition of PS2.M parallel quadruplex, the hemin dissociation constant (Kd) of PS2.M, and pKa of PS2.M/hemin are summarized in Table 2. The Tm of PS2.M alone in 100 mM KCl was 52 °C and the Tm of PS2.M in the presence of PLL-g-Dex was increased to over 80 °C, indicating a remarkable effect of PLL-g-Dex on parallel quadruplex stabillization. In 100 mM NaCl, the Tm of PS2.M in parallel structure could not be determined and that of anti-parallel structure was determined to be 49 °C. In 100 mM NaCl and in the presence of PLL-g-Dex, the Tm of PS2.M in parallel structure was 61 °C (CD melting curves shown as Figure S7 in Supporting Information). These results indicated that PLL-g-Dex selectively stabilized the parallel G-quadruplex structure over antiparallel one in not only K+ buffer but also Na+ buffer. The phosphate groups of DNA were more densely arranged around an intra-molecular quadruplex core with parallel folding


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than that with antiparallel folding.

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PLL-g-Dex effectively shielded electrostatic repulsions

among the phosphate groups to stabilize the parallel structure. Both Tm and kcat increased in the presence of PLL-g-Dex, indicating that stabilization of parallel G-quadruplex structure was involved in the enhancement mechanisms of the DNAzyme catalytic activity. Affinity of hemin to PS2.M in the absence and presence of PLL-g-Dex was determined by absorption titration experiments.21 In 100 mM KCl, the Kd values of PS2.M for hemin without and with of PLL-g-Dex were 13 nM and 41 nM, respectively (Table 2). Since PLL-gDex has abundant dextran side chains, hemin binding to PS2.M could be reduced sterically. The Kd values in 100 mM KCl are, however, enough lower than the hemin concentration (0.5 µM) used in the catalytic activity measurements in this study, so that PS2.M/hemin complex formation was saturated (fraction of hemin existing as PS2.M/hemin complex calculated on the basis of the Kd values was shown in Figure S8 in the Supporing Information). Thus, the increase in Kd by the copolymer did not cause a loss in enzymatic activity under our experimental conditions. Meanwhile the DNAzyme activity was enhanced by parallel quadruplex stabilization by the copolymer. Beside the parallel quadruplex stabilization effect the copolymer would increase DNAzyme activity by modifying hemin/quadruplex interactions. Travascio and coworkers reported that ribonucleic acid version (rPS2.M) of PS2.M shows higher activity than PS2.M, while the binding affinity of hemin to rPS2.M is weaker than that to PS2.M.29 In 100 mM NaCl, the Kd of PS2.M for hemin/PS2.M complex in the absence of PLL-gDex was considerably higher than that in 100 mM KCl, indicating weaker interaction of hemin to antiparallel PS2.M. As a structural transition from antiparallel G-quadruplex to parallel Gquadruplex is induced by PLL-g-Dex, hemin binds more tightly in the presence of PLL-g-Dex, leading to 30-time enhancement of the DNAzyme activity. It was possible to further increasing


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the DNAzyme activity by increasing PS2.M concentration (Figure 5(b)), resulting in similar kcat value that observed in 100 mM KCl with PLL-g-Dex. These kcat values obtained for PS2.M DNAzyme in the presence of PLL-g-Dex are higher than those reported previously including the attempts to enhance the catalytic activity by DNA modifications such as 2’-o-methyl and by the presence of a cationic peptide.26,27 It was recently reported that pKa value of hemin affected DNAzyme activity.30 The effect of PLL-g-Dex on pKa of hemin/PS2.M complex was then investigated by spectrophotometric titration (titration curves shown as Figure S9 in Supporting Information) and the results were shown in Table 2. In 100 mM KCl, the pKa value in the absence of PLL-g-Dex was higher than that in the presence. In 100 mM NaCl, however, the pKa value in the absence PLL-g-Dex was lower than that in the presence. In either NaCl or KCl buffer, enzymic activity of PS2.M/hemin was enhanced by the presence of PLL-g-Dex. These results implied that the enhancement effect of PLL-g-Dex was not linked to hemin pKa. In enhancement of the activity, other factors in addition to stabilization of parallel G-quadruplex would also exist.

Canale and co-worker

reported that hemin/G-quadruplex DNAzyme is enhanced in water-methanol mixed solvents compared with water,31 suggesting that the environment around DNAzyme is important in the catalytic activity. PLL-g-Dex could change micro environmental factors, topology of hemin binding and parallel G4 structure in molecular level. In these ways, higher activity of the DNAzyme was successfully achieved in the presence of PLL-g-Dex.


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Figure 5. DNA concentration dependence of DNAzyme reaction in the absence or presence of PLL-g-Dex in (a) 100 mM KCl or (b) 100 mM NaCl in 25 mM NaH2PO4/Na2HPO4 (pH 7.0), 0.5 µM hemin, 100 µM ABTS, 200 µM H2O2, PLL-g -Dex (N/P ratio of 0 or 10) and PS2.M at 0.5 µM (white bars), 1.0 µM (grey bars), or 1.5 µM (black bars).

3.3 Reversible allosteric control of the DNAzyme activity by PLL-g-Dex Finally, we demonstrated reversible activation of the DNAzyme using cationic and anionic polymers as effectors (Figure 6(a)). PLL-g-Dex, the positive effector, stabilizes the parallel G-quadruplex, and the DNAzyme forms the active state. An anionic polymer, PVS, acts as a neutralizer of PLL-g-Dex. The anionic polymer induces dissociation of PLL-g-Dex from PS2.M, and the DNAzyme returns to the inactive state. During the enzyme reaction, PLL-g-Dex and PVS were added successively several times (Figure 6(b)). In the initial state, absorbance at 414 nm slightly increased over time. After addition of PLL-g-Dex, a rapid increase in the absorbance derived from ABTS was observed. When 1.5-fold excess (relative to added PLL-gDex charge) PVS was added to reaction solution, the increase in absorbance at 414 nm slowed. The Vobs values of initial state and after addition of PVS were in the range of 5-20 nM/sec,


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whereas Vobs was more than 80 nM/sec in the presence of PLL-g-Dex (Figure 6(c)). Thus, reversible allosteric control of DNAzyme activity was successfully carried out by an electrostatically small excess of polycations and polyanions. To the best of our knowledge, this is the first achievement of allosteric-based reversible activation-deactivation of a DNAzyme using artificial polymer materials.

Figure 6. Allosteric control of DNAzyme activity with cationic and anionic polymers. (a) Schematic illustration of the reaction. (b) Absorbance change at 414 nm upon sequential addition of PLL-g-Dex and PVS. (c) Vobs of the DNAzyme reaction. Conditions: 25 mM NaH2PO4/Na2HPO4 (pH 7.0), 100 mM NaCl, 1.0 µM PS2.M, 0.5 µM hemin, 100 µM ABTS, 200 µM H2O2, and PLL-g-Dex (N/P ratio of 0 or 10).


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4. CONCLUSIONS In this study, we showed that peroxidase-mimicking DNAzyme catalytic activity was successfully enhanced by the cationic comb-type copolymer, PLL-g-Dex. PLL-g-Dex stabilized parallel G-quadruplex structures, which have higher DNAzyme activity than other G-quadruplex structures in both K+ and Na+. Stabilization of the parallel G-quadruplex by PLL-g-Dex was explained based on CD structure and stability analyses. For example, the Tm of PS2.M was increased by more than 30 oC in the presence of PLL-g-Dex relative to its absence. Stabilization of parallel G-quadruplex structures with PLL-g-Dex promoted the binding of hemin to PS2.M enabling catalysis in 100 mM NaCl. Moreover, reversible allosteric control of DNAzyme activity was achieved using sequential addition of PLL-g-Dex and an anionic polymer. Allosteric effect of the copolymer toward DNA quadruplexes will find utility in other quadruplex-based molecular devices for biosensing and molecular robotics.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Effect of PLL-g-Dex on the DNAzyme reaction, oxidization of tryptophan by the DNAzyme, CD spectrum of PLL-g-Dex alone, CD spectra of HT and G8, CD-Tm curves of PS2.M, the Vobs of PS2.M against different concentrations of H2O2 or DNA, and the titration curve for PS2.M/hemin (PDF).


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AUTHOR INFORMATION Corresponding Author *E-mail: [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 Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT This work was supported in part by Center of Innovation (COI), Japan Science and Technology Agency (JST) and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant No. 15H01807 to A.M.).

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Table of Contents (TOC)


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Allosteric control of peroxidase-mimicking DNAzyme activity with

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