Cooperative enhancement of deoxyribozyme activity by chemical modification and added cationic copolymer

Abstract Deoxyribozymes (DNAzymes) having RNA-cleaving activity have widely been explored as tools for therapeutic and diagnostic purposes. Both the chemical cleaving step and the turnover step should be improved for enhancing overall activity of DNAzymes. We have shown that cationic copolymer enhanced DNAzyme activity by increasing turnover efficacy. In this paper, effectｓ of the copolymer on DNAzymes modified with locked nucleic acids (LNA) or 2′-O-methylated (2′-OMe) nucleic acids were studied. The copolymer increased activity of these chemically modified DNAzymes. More than 30-fold enhancement in multiple-turnover catalytic activity was observed with 2′-OMe-modified DNAzyme in the presence of the copolymer. DNAzyme catalytic activity was successfully enhanced by cooperation of the added copolymer and chemical modification of DNAzyme.


Introduction
Deoxyribozymes (DNAzymes) are DNA sequences having catalytic activities. [1][2][3] DNAzymes have several advantages over ribozymes or protein enzymes. DNAzymes are chemically and biologically more stable than ribozymes and protein enzymes, allowing ease in handling and storage. DNAzymes are readily prepared and modified. The 10-23 DNAzyme has catalytic activity to cleave RNA with sequence-specific manner. [4][5][6][7] Its activity exceeds that of any other known nucleic acid enzymes. 10-23 DNAzyme has been extensively studied for biomedical applications, such as nucleic acid medicines and genetic analyses. [8][9][10][11][12] However, the catalytic activity should be improved to satisfy these applications.
We have reported that cationic comb-type copolymers, PLL-g-Dex, consisting of a polycationic backbone and water soluble grafts facilitated hybridization of nucleic acids by reducing electrostatic repulsion between nucleic acid strands. [13][14][15][16][17] We recently reported that the copolymer accelerated multiple-turnover reactivity of 10-23 DNAzyme [18] and its derivative, multicomponent nucleotide enzyme (MNAzyme). [19] A variety of chemically modified nucleic acids was explored to improve hybridization properties of nucleic acids. Locked nucleic acid (LNA), [20,21] and 2′-O-methylated (2′-OMe) nucleic acids [22,23] convey an RNA-like character to the DNA strands inserted with these modified nucleic acids. Enhanced activity of DNAzyme by insertion of chemically modified nucleic acids to substrate-binding arms of DNAzyme was reported. [24][25][26] To improve further the DNAzyme reactivity, we are interested in examining the cooperation of the added cationic comb-type copolymer in the reaction of the chemically modified DNAzymes. In this study, catalytic activities of 10-23 DNAzyme modified with LNA or 2′-OMe were evaluated in the absence and presence of PLL-g-Dex. While 2′-OMe modification and the copolymer showed 1.5-fold and 17-fold enhancements, respectively, 30-fold enhancement of DNAzyme activity by this modification was observed in the presence of the copolymer. The result clearly demonstrated that successful cooperation of the added copolymer and chemical modification of DNAzyme.

Förster resonance energy transfer analysis to trace the DNAzyme cleavage reaction
A substrate labeled with fluorescein isothiocyanate (FITC) and BHQ-1 quencher (final concentration: 200 nM) in 50 mM HEPES (pH 7.3), 150 mM NaCl, and 5.0 mM Mn 2+ was pre-incubated with PLL-g-Dex at the ratio of [positively charged amino groups] copolymer /[negatively charged phosphate groups] DNA (N/P ratio) of 2 at the reaction temperature for 5 min in a quartz cell. DNAzyme reaction was initiated by injecting DNAzyme solution (final concentration: 0.25 or 2.0 nM) into the cell. The fluorescence intensity of the solution was acquired using a fluorescence spectrometer (FP-6500 Jasco, Tokyo, Japan) at an excitation wavelength, λ ex , of 494 nm and an emission wavelength, λ em , of 520 nm, respectively, with excitation and emission slits at 3 nm. The fluorescence intensity curve over time was used to fit the following equation.
where I t was the fluorescence intensity at any reaction time t, I ∞ was the fluorescence intensity after incubating at 50°C for 24 h, I 0 was the initial fluorescence intensity (background).
Initial period of the cleavage reaction curve was fitted to estimate the k obs values.

Melting temperature measurement
DNAzyme and substrate (1.0 μM in final concentration) were mixed in a 50 mM HEPES containing 150 mM NaCl (pH 7.3) in the absence or presence of PLL-g-Dex (N/P = 2). The mixture was heated at 95°C for 5 min, followed by subsequent slow cooling to room temperature over 12 h. UV-T m curves were obtained at 260 nm on V-630 spectrophotometer (Jasco) at heating rate of 1.0°C min -1 from 25 to 95°C. Melting data were collected and fitted with Spectra Manager (Jasco).

Results and discussion
We observed DNAzyme reaction in the absence or presence PLL-g-Dex at 35°C for 30 min under multiple-turnover condition, [S] / [E] = 50. The results are shown in Figure 3. While LNA modification significantly decreased DNAzyme reaction rate, 2′-OMe modification increased it.
As reported previously, the cationic comb-type copolymer increased the reaction rate of unmodified DNAzyme. Similar accelerating effect of the copolymer was observed for 2′-OMe modified DNAzyme. The accelerating effect of the copolymer on LNA-modified DNAzyme was also observed but its effect was not considerable compared to those observed with unmodified or the LNA-modified one.
Chemical modification of DNAzyme at its substrate binding arms influences stability of enzyme/product complex and enzyme/substrate complex, so that the optimum temperatures for DNAzyme reactions should be influenced by the modification. To estimate influence of the chemical modification on the optimum temperatures, temperature dependences of DNAzyme activities were estimated. As shown in Figure 4, the unmodified DNAzyme has an optimum temperature at 50°C. While the 2′-OMe modification did not significantly alter the optimum temperature it increased the DNAzyme activity. The LNA-modified DNAzyme has an optimum temperature at 60°C, indicating significant increase in the optimum temperature. The LNA-modified DNAzyme activity was reduced. We then estimated stability of enzyme-substrate (ES) complexes by measuring their melting temperatures, T m s. UV-melting curves of ES complex are shown in Figure 5. The values of T m determined from Figure 5 and the reaction rates of DNAzymes at the optimum temperatures are summarized in Table 1. Close relationships between the optimum temperature and the T m values were shown. The results indicated that DNAzyme activity was not determined by the chemical cleavage step (k 2 in Scheme 1) but hybridization dynamics (k 1 and k 3 and their inverse reaction rates in Scheme 1) of DNAzymes with either its substrates or products (P). In general, with increasing temperature, dissociation rates of DNA hybrids increase while their association rates decrease. The increase in enzymatic activity with increasing temperature up to the optimum temperatures is attributed to the increase in dissociation rate of EP complex. Decrease in the enzymatic activity at temperatures higher than the optimum temperature resulted from a decrease in association rate of the ES complex. We observed a 10°C increase in T m for the ES complex with LNA modification. It is well established that that LNA modification increased stability of DNA hybrids. [20,21] In our experimental conditions, LNA modification resulted in approximately 40% loss in the DNAzyme activity. LNA modification stabilized enzyme/product (EP) complex as well as ES complex and reduced dissociation rate (k 3 in Scheme 1) of the   2′-OMe modification are nearly consistent with the product of 1.5-fold and 17.5-fold enhancements that were observed for 2′-Me modification and the added copolymer, respectively. The copolymer was also effective to enhance activity of LNA-modified DNAzyme, although the magnitude of the enhancement is moderate. LNA-modified DNAzyme showed considerably stronger activity than the unmodified one when substrates had intramolecular structures. [28] Strong EP complex. The decrease in the dissociation rate of the EP complex resulted in slow turnover, leading to the decrease in DNAzyme reactivity under multiple turnover conditions. This consideration was supported by the fact that the LNA modification stabilizes the DNA hybrid by reducing the dissociation rate rather than increasing the association rate of hybridization. [27] LNA modification was considered to positively affect the DNAzyme reactivity by stabilizing the ES complex (i.e. decreasing Michaelis constant K M ) if the reaction is carried out under single-turnover reaction condition. While 2′-OMe modification resulted in a slight decrease in T m , it increased 1.5 times the catalytic activity compared to that of unmodified DNAzyme when compared at their optimum temperatures. We speculate that 2′-OMe modification decreased T m by increasing the dissociation rate of the DNA duplex. The 2′-OMe modification seemingly promoted dissociation of EP complex, resulted in faster multiple-turnover reaction. We then assessed the influence of added PLL-g-Dex copolymers on unmodified and chemically modified DNAzymes ( Figure 6 and Table 1). The copolymer increased more than 17-fold the catalytic activity of DNAzymes at the optimum temperature, in accordance with our previous observation. [18] The copolymer increased T m of the ES complex by 11.5°C. The added PLL-g-Dex increased T m of the ES complex similarly to LNA-modification, indicating both the added PLL-g-Dex and the LNA modification contributed to the stabilization of ES complex. Stability of EP complex should also be increased by the added PLL-g-Dex as well as the LNA modification. In contrast to the LNA modification that stabilizes the DNA hybrid by decreasing a dissociation rate of a DNA hybrid, the copolymer does it principally by increasing its association. The copolymer likely increases the turnover step by promoting ES complex formation whereas the LNA modification retards it by decreasing the dissociation rate of the EP complex.
It is noted that the highest catalytic activity was observed with 2′-OMe modification in the presence of the copolymer. More than 30-fold enhancement in multiple-turnover reactivity was found. The added copolymer and 2′-OMe modification contributed the diverse steps, i.e. k 1 and k 3 , respectively, in the turnover process, so that their activities are well harmonized. In fact, the 30-fold enhancement achieved by the copolymer and  cationic comb-type copolymer, PLL-g-Dex, increased multiple-turnover reactivity of chemically modified DNAzymes as well as an unmodified DNAzyme. Thirtyfold enhancement of the activity was achieved with 2′-OMe modification in the presence of the copolymer. Kinetic effects rather than thermodynamic effect of the chemical modifications and the copolymer on DNA hybridization likely play a pivotal role in the observed cooperative effect.

Disclosure statement
No potential conflict of interest was reported by the authors. hybridization ability of LNA-modified binding arms permitted invasion of the DNAzyme to the structured substrates. Our result suggested that the copolymer in combination with LNA modification would be useful to enhance DNAzyme activity toward structured targets. Our previous study indicated that the copolymer further enhanced hybridization property of LNA modified oligonucleotide. [29] The copolymer promoted DNA/LNA assembly as well as DNA/DNA assembly by reducing the counterion condensation effect as estimated by thermodynamic analysis. [29] As seen from Figure 5, the copolymer considerably increased enzymatic activity of DNAzymes examined over a wide temperature range. Below the optimum temperature, the rate-determining step is the dissociation of the EP complex, so that the copolymer was thought not to be effective to accelerate the reaction by promoting ES complex formation. Nevertheless, obvious accelerating effects of the copolymer were observed even at a temperature significantly lower than the optimum temperature. It would be possible for the copolymer to facilitate a turnover process by the strand exchange pathway (k 4 of Scheme 1) of EP complexes with substrates to form fresh ES complex, because the copolymer is capable of activating strand exchange reactions between double stranded DNA and its homologous single strand DNA. [30,31]

Conclusions
In this study, we showed that DNAzyme catalytic activity was successfully enhanced by cooperation of the added copolymer and chemical modification of DNAzyme. The Scheme 1. Schematic representation of dnazyme reaction pathways.