Cationic copolymer-chaperoned DNAzyme sensor for microRNA detection

Biomaterials 225 (2019) 119535

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Cationic copolymer-chaperoned DNAzyme sensor for microRNA detection Orakan Hanpanich, Tomoya Oyanagi, Naohiko Shimada, Atsushi Maruyama

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T

Department of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta 4259 B-57, Yokohama, 226-8501, Japan

A R T I C LE I N FO

A B S T R A C T

Keywords: Allosteric DNAzyme Cationic copolymer microRNA Biosensing Artificial chaperone Molecular assembly

Multi-component nucleic acid enzymes (MNAzymes) are allosteric deoxyribozymes that are activated upon binding of a specific nucleic acid effector. MNAzyme activity is limited due to an insufficient assembly of the MNAzyme and its turnover. In this work, we describe the successful improvement of MNAzyme reactivity and selectivity by addition of cationic copolymers, which exhibit nucleic acid chaperone-like activity. The copolymer allowed a 210-fold increase in signal activity and a 95-fold increase in the signal-to-background selectivity of MNAzymes constructed for microRNA (miRNA) detection. The selectivity of the MNAzyme for homologous miRNAs was demonstrated in a multiplex format in which isothermal reactions of two different MNAzymes were performed. In addition, the copolymer permitted miRNA detections even in the presence of a ribonuclease which is ubiquitous in environments, indicating the protective effect of the copolymer against ribonucleases.

1. Introduction Deoxyribozymes (DNAzymes) are catalytically active DNA molecules [1] providing several advantages compared to protein-based enzymes such as high stability [2,3] and ease of synthesis. The RNAcleaving DNAzymes consists of a catalytic core region flanked by two substrate-binding arms (S-arms) that can be tailored to bind any sequence through Watson-Crick base pairing. Due to their programmable characteristics, DNAzymes have potential for use in biosensors and nanotechnology applications [4–9]. The catalytic core of DNAzymes can be split into two fragments and conjugated with effector recognition arms (E-arms) to create allosterically controlled multi-component nucleic acid enzymes (MNAzymes) (Fig. 1a) [10]. MNAzymes are activated upon binding of the specific effector nucleic acid, which reunites the partial catalytic core regions and recovers the catalytic activity. The active MNAzymes are able to cleave multiple substrates and amplify the signal corresponding to the effector. Advantages of MNAzymes are the high specificity of substrate and effector recognition due to base pairing; isothermal and enzyme-free amplification; generality; and simplicity. MNAzymes have been used as biosensors and as components of nanomachines and molecular computers [10–15]. MNAzyme reaction involves MNAzyme association, catalysis of phosphodiester bond cleavage, and product dissociation (Scheme 1a). Strategies to improve the cleavage step have been reported such as optimization of the metal ion cofactors [16–18] and substitution of deoxyinosine at the cleavage site [18]. Efforts have been made to promote association by improving hybridization properties of nucleic

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acids, such as the introduction of 2′-O-methylated nucleotides [19,20], and locked nucleic acid residues [19–21], but substitution of modified nucleotides within the nucleic acid enzyme structure often leads to conformational change that reduces catalytic activity [22]. Moreover, an improvement of substrate turnover is limited because of the compromise between substrate association and product dissociation. For example, changes in sequence that enhance association typically reduce the rate of product dissociation [19,20], and, although the dissociation rate can be accelerated by increasing the reaction temperature, this decreases the association rate. Insufficient assembly of MNAzyme with short nucleotide effectors, such as microRNA (miRNA), is another issue. When the MNAzyme-effector complex is thermodynamically unstable, the activity of the MNAzyme is low, especially under multiple-turnover conditions. Another concern is the cleavage reaction in the absence of target nucleotide effectors. This background reaction causes false-positive signals when the MNAzyme is utilized as a sensor. Hence methods to enhance both catalytic activity and selectivity are needed. Our group has used cationic copolymers grafted with hydrophilic side chains, the poly(L-lysine)-graft-dextran (PLL-g-Dex) (Fig. 1b), to stabilize DNA duplex [24,25], triplex [25–28], and quadruplex [29–31] structures for various applications. The copolymer accelerates DNA hybridization [28] and DNA strand-exchange reactions [32–34], implicating that the copolymer produces nucleic acid chaperone-like activity. These properties are preferable for nucleic acid-based nanotechnology applications such as photo-driven strand displacement reactions [35], molecular beacon probes [36], DNA-fueled nanomachines [37], DNA logic gates [38], DNAzymes [39], and MNAzyme

Corresponding author. E-mail address: [email protected] (A. Maruyama).

https://doi.org/10.1016/j.biomaterials.2019.119535 Received 18 June 2019; Received in revised form 1 October 2019; Accepted 2 October 2019 Available online 05 October 2019 0142-9612/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. MNAzyme activities at various temperatures. (a) Sequences of MNAzyme fragments. Lowercase letters denote RNA embedded in the substrate as a cleavage site, letters F and Q denote the fluorescence donor and the quencher, respectively. X and Y indicate S-arm lengths in nucleotides (nt) (b) Chemical structure of PLL-g-Dex. Percent substrate cleavage with time by MNAzyme Mz(10,11) in (c) the absence and (d) the presence of PLL-g-Dex (molar ratio of cationic amino groups of the copolymer to negatively charged phosphate groups of nucleotides (N/P) was 2). (e) Reaction rate (kobs) at various temperatures in the absence (dashed line, open symbol) and the presence (solid line, close symbol) of PLL-g-Dex. Experimental conditions: 50 mM HEPES, 150 mM NaCl, pH 7.3, 5 mM MnCl2, 100 nM substrate, 20 nM enzyme, and 20 nM miR-21 effector without or with PLL-g-Dex.

Mn = 8.0 × 103–1.2 × 104) was obtained from Funakoshi Co. (Tokyo, Japan). Sodium hydroxide, sodium chloride, and manganese (II) chloride tetrahydrate were purchased from Wako Pure Chemical Industries (Osaka, Japan). 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Oligonucleotides used in this study were purchased from Fasmac Co., Ltd (Atsugi, Japan); oligonucleotides were HPLC-grade and were used without further purification.

[23]. Recently, we reported that the copolymer considerably enhanced catalytic activity of an MNAzyme with long effector arms (29 nucleotides). In this work, we simultaneously enhanced selectivity and activity of MNAzymes having short effector arms (22 nucleotides) by addition of PLL-g-Dex to the reaction buffer. Short nucleic acids such as miRNAs are potential biomarkers of diverse diseases. However, the sensitivity and selectivity of assays designed to detect miRNAs are generally limited by the intrinsic properties of the miRNAs including short lengths, low stability, low abundance, and high sequence homology among family members [40]. The copolymer promoted the MNAzyme assembly and increased turnover of substrates and/or effectors. Moreover, the copolymer allowed S-arm truncation which reduced target-independent background without loss of target-dependent catalytic efficiency. The copolymer allows simple, rapid, and quantitative detection of miRNA with sensitivity comparable to that of the commercially available miRNA detection kits based-on qRT-PCR. The MNAzyme reaction can be performed in mild conditions in one pot without sophisticated instruments and does not require labor-intensive steps. These features make MNAzyme-based nanodevices and diagnostic strategies more attractive. Finally, the copolymer also protected RNA from the proteinous ribonuclease, RNase A, suggesting that the MNAzyme-based detection of miRNA can be performed in cell extracts.

2.2. Synthesis of poly(L-lysine)-graft-dextran Poly(L-lysine)-graft-dextran (PLL-g-Dex) consisted of 10 wt% PLL and 90 wt% dextran (11.5 mol.% of lysine units of PLL were substituted with dextran) was prepared according to the previously published protocol [26]. Briefly, PLL-g-Dex was obtained by reductive amination reaction of dextran with PLL. The polymers were purified over an ionexchange column, dialyzed, and freeze-dried. The products were then characterized by 1H NMR (Bruker Avance 400) at 60 °C and by GPC. 2.3. MNAzyme constructs The catalytic core of the MNAzyme was derived from the DNAzyme 10–23 as described by Mokany et al. [10]. Each MNAzyme subunit (partzyme) consists of a substrate-binding arm (S-arm), a partial catalytic core, and an effector binding arm (E-arm) (Fig. 1a). The MNAzyme nomenclature used here is Mz(X,Y), where X and Y are S-arm lengths in nucleotides (nt) of 5′-arm and 3′-arm, respectively. The cleavable substrate is a single-stranded DNA/RNA chimera. An RNA 5′-GU-3′ is embedded in an otherwise deoxyoligonucleotide where cleavage occurs

2. Materials and methods 2.1. Materials Poly(L-lysine hydrobromide) (Mw = 7.5 × 103) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dextran (Dex, 2

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It = I0 + (I∞ − I0) (1 − e−k obs t ) 2.5. Thermal denaturation analysis Samples for thermal denaturation measurements contained 50 nM substrate, 50 nM enzyme, and 50 nM effector in 50 mM HEPES, 150 mM NaCl, pH 7.3 in the presence (N/P of 2) or absence of PLL-g-Dex. In these experiments, the substrate strands were all DNA instead of the cleavable DNA/RNA chimera. MNAzyme constructs used in this experiment was shown in Figs. 3a, 3b, S6a and S6b. The mixture was heated from 5°C to 80°C at a rate of 1 °C/min. The fluorescence intensity was recorded using an FP-6500 spectrofluorometer (Jasco, Tokyo, Japan) with excitation and emission wavelength 494 nm and 520 nm, respectively. 2.6. Analysis of protective effect against RNase degradation The effector miR-21 (20 nM or 100 nM in final concentration) was dissolved in a 50 mM HEPES containing 150 mM NaCl (pH 7.3). The mixtures were incubated with 100 ng/mL of RNase A for 30 min at 37 °C. PLL-g-Dex (N/P = 5) was either added to the mixture before or after incubation with ribonuclease A (RNase A). Following this, substrate and partzymes were added to the mixtures. The concentration of substrate and enzyme were 100 nM and 20 nM, respectively. MNAzyme reactions were initiated by adding MnCl2 solution (5 mM in final concentration). Fluorescence intensity was measured and MNAzyme activities were analyzed with the same method as mentioned before.

Scheme 1. MNAzyme reaction scheme. (a) effector-dependent (signal) and (b) effector-independent (background) reactions. MNAzyme subunits (partzymes) binds to its substrate and effector to form active structure. Subsequently, the MNAzyme catalyzes the substrate cleavage. Background reaction occurs when partzymes bind and cleave substrate without effector binding.

3. Results 3.1. Enhancement of effector-dependent reactivity by PLL-g-Dex Small non-coding miRNAs play a crucial role in various cellular functions, and associate with progression of diverse diseases including cardiovascular disease, neurodegenerative disease and cancers [41–43]. Although miRNAs are considered as a potential and minimally-invasive biomarker, their intrinsic properties including short lengths, low stability, low abundant, and high sequence homology among family members limit sensitivity and selectivity of the detection [40]. Activities of the MNAzyme, Mz(10,11), were evaluated under multiple turnover condition at different temperature to compare activities at the optimum temperatures. The effector used in this study is a well-known disease-related miRNA-21 (miR-21) [44]. Product formation in the presence of 20 nM miR-21 effector was slow owing to an insufficient assembly of active MNAzyme complex composed of a substrate, enzyme components (partzymes), and an effector. Significant acceleration of product formation was observed in the presence of the cationic copolymer (Fig. 1c and d). Reaction rates (kobs) determined at different temperatures in the presence or absence of PLL-g-Dex (Fig. 1e) indicated that the optimal temperature was increased from 35°C to 50°C and reaction rate was increased by two orders of magnitude from 4.98 × 10−3 min−1 to 3.91 × 10−1 min−1 by addition of PLL-g-Dex. Reaction rate observed in the presence of the copolymer under the multiple turnover conditions ([Enzyme] = [Effector] = 20 nM and [substrate] = 100 nM) of Mz(10,11) was comparable to that observed with the MNAzyme having a longer effector (29 nt), Mz(10,11)L, under the single turnover conditions ([Enzyme] = [Effector] = [substrate] = 100 nM) without the copolymer (Fig. S1) at 40 °C, which was the optimum temperature for the latter. These findings strongly suggested that the copolymer not only promoted the MNAzyme assembly with a short miRNA effector but also increased turnovers of substrates and/or effectors. Further increase in the reaction rate of Mz(10,11) was attained at 50 °C under the multiple turnover conditions in the presence of the copolymer (Fig. 1d). Signal-to-background selectivity (S/B) of MNAzyme was evaluated

between the two ribonucleotides. The substrate was labeled with fluorescein isothiocyanate (FITC) and black hole quencher 1 (BHQ-1), or with carboxytetramethylrhodamine (TAMRA) and BHQ-2. The fluorescent donor was conjugated at the 5′ of the cleavage site, and the quencher was conjugated at the 3′ terminus.

2.4. MNAzyme reaction analysis Cleavage of the substrate by the MNAzyme was detected by Förster Resonance Energy Transfer (FRET) analysis. Unless otherwise indicated, 100 nM substrate, 20 nM enzyme, and 20 nM effector were preincubated in 50 mM HEPES, 150 mM NaCl, pH 7.3 at the indicated reaction temperature. After 5 min, either PLL-g-Dex or buffer was injected into the reaction solution. The molar ratio of cationic amino groups of the copolymer to negatively charged phosphate groups of nucleotides (N/P) in the final solution was two. The MNAzyme reactions were initiated by injection of MnCl2 solution to a final concentration of 5 mM, at 90 s after PLL-g-Dex injection. The cleavage reaction separated the fluorescent donor from the quencher resulting in increased fluorescence over time, which was measured using an FP-6500 spectrofluorometer (Jasco, Tokyo, Japan). Excitation and emission wavelengths were 494 nm and 520 nm, respectively (or 556 nm and 580 nm for the substrate labeled with TAMRA and BHQ-2). Excitation and emission slits (bandwidth) were set at 3 nm. The percent substrate cleavage over time was obtained from the following equation:

substrate cleavage (%) = [(It − I0)/(I∞ − I0)] × 100 where It is the fluorescence intensity at a given reaction time t, I∞ is the fluorescence intensity after incubating the reaction until saturation, and I0 is the initial fluorescence intensity. The values of kobs were calculated by fitting the reaction curve to the following equation: 3

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Table 1 Optimal temperatures, reaction rates, and signal-to-background ratios of MNAzymes with different S-arm lengths.a. MNAzyme

PLL-g-Dex

Optimal temp. (°C)

kobs (min−1)

S/B ratio

miR-21 (+) Mz(10,11) Mz(7,8) Mz(6,7) Mz(5,6)

– + – + – + – +

35 50 25 37 20 30 15 25

−3

4.98 × 10 3.91 × 10−1 4.64 × 10−3 1.05 (210) 2.08 × 10−3 9.42 × 10−1 2.25 × 10−4 2.49 × 10−1

miR-21 (−) b

(1.0) (79) (0.93) (0.42) (190) (0.045) (50)

1.54 × 10−4 6.54 × 10−3 5.80 × 10−5 7.14 × 10−4 7.26 × 10−6 3.06 × 10−4 6.72 × 10−6 2.21 × 10−4

(1.0) (43) (0.38) (4.6) (0.047) (2.0) (0.044) (1.4)

32.3 (1.0) 59.7 (1.9) 80.0 (2.5) 1.47 × 103 (46) 286 (8.9) 3.08 × 103 (95) 33.5 (1.0) 1.13 × 103 (35)

a Experimental conditions: 50 mM HEPES, 150 mM NaCl, pH 7.3, 5 mM MnCl2, 100 nM substrate, 20 nM enzyme, and either 0 or 20 nM miR-21 effector, without or with PLL-g-Dex (N/P of 2). b The number in parenthesis indicates ratio relative to Mz(10,11) in the absence of PLL-g-Dex.

Dex without truncation approximately increased S/B by 2 fold and Sarm truncation in the absence of PLL-g-Dex increased S/B maximally 9 fold. The combination of the most effective S-arm truncation and the addition of PLL-g-Dex increased S/B up to 95 fold (Table 1). While PLLg-Dex facilitated substrate association with the MNAzyme (k1), the shorter S-arms promoted product dissociation (k3). The combination of these events successfully overcame the association-dissociation conflict and synergistically accelerating signal reaction rate by promoting turnover.

at the optimal temperatures. The ratio of reaction rate in the presence and the absence of the miR-21 effector (signal to background (S/B) ratio) of Mz(10,11) without PLL-g-Dex was 32.3 (Fig. S2a and Table 1). In the presence of PLL-g-Dex, the S/B ratio was increased to 59.7 (Fig. S2b and Table 1). Although the effector-dependent reactivity (signal) (Scheme 1a) was significantly accelerated by PLL-g-Dex, undesired effector-independent reactivity (background) (Scheme 1b) remained significant and the improvement of the S/B ratio was limited. In the absence of PLL-g-Dex, relatively high background (6.57 × 10−4 min−1) was observed at 30°C. Background decreased (6.18 × 10−5 min−1) when the temperature increased to 40 °C as enzyme-substrate complexes were destabilized. Above 40°C, spontaneous hydrolysis of the substrate became the major cause of background (Fig. S3a). Similar trends were observed in the presence of PLL-g-Dex, but the enzymesubstrate complexes were destabilized at higher temperature. Effectorindependent reactivity due to enzyme-substrate assembly was eliminated at 60°C (Fig. S3b), while at 40°C in the absence of the copolymer (Fig. S3a).

3.3. Thermal denaturation study of MNAzyme complexes The thermal stability of MNAzyme complex was determined by melting curve analyses to understand the mechanism underlying the effect of PLL-g-Dex. To prevent substrate cleavage during analysis, we used a non-cleavable substrate strand, in which the two RNA residues were replaced with DNA counterparts. To monitor thermal dissociation of the Mz(10,11) and Mz(6,7) complexes, the quencher BHQ-1 was linked to the 3′-end of the partzyme and the fluorescence donor FITC was conjugated to the non-cleavable substrate strand (Fig. 3a and b). The plot of fluorescence intensity versus temperature of Mz(10,11) in the absence of PLL-g-Dex revealed that between 5°C and 40°C, the hybridization of substrate-partzymes resulted in relatively weak fluorescence intensity compared to the free substrate because fluorophore and quencher were close to one another in the MNAzyme complex (Fig. 3c). The complex began to dissociate when the sample was heated above 40°C. Separation of a donor-quencher pair increased fluorescence intensity within experimental error of the fluorescence intensity of the free substrate at 60°C. The melting curve of Mz(10,11) in the presence of PLL-g-Dex was shifted to higher temperature by 20°C (Fig. 3d) consistent with the higher optimal cleavage reaction temperature in the presence of the copolymer (Fig. 1e). For the truncated MNAzyme Mz (6,7) in the absence of PLL-g-Dex, even at temperatures below 30 °C, there was little complex formation (Fig. 3e). In the presence of PLL-gDex, the substrate-Mz(6,7) association was significantly enhanced as a clear melting transition was observed (Fig. 3f). Extremely low stability of short-armed Mz(6,7) correlated with low reaction rate (Fig. 3e, Table 1). On the contrary, higher stability in the presence of PLL-g-Dex correlated with increasing cleavage activity for both Mz(10,11) and Mz(6,7) (Fig. 3d and f, and Table 1). In the presence of PLL-g-Dex, short-armed Mz(6,7) showed lower stability but exhibited higher cleavage activity than long-armed Mz(10,11). The results suggested that the effect of the copolymer sufficiently promoted substrate association (k1) and activity of long-armed MNAzyme might be limited by the dissociation of products (k3). Therefore, S-arm truncation further increased MNAzyme activity because the product dissociation (k3) was promoted. The additional S-arms truncation eventually resulted in decreased the activity of Mz(5,6) lower than that of Mz(10,11). Under this condition, it is assumed that the activities of

3.2. The combination of S-arms truncation and PLL-g-Dex improves reactivity and selectivity To reduce effector-independent background reaction, substratebinding arms (S-arms) of Mz(10,11) were truncated to destabilize enzyme-substrate complex in the absence of the effector. The truncated MNAzymes, Mz(7,8), Mz(6,7), and Mz(5,6), were newly synthesized (MNAzyme sequences are shown in Fig. S4). The melting temperature (Tm) of S-arms calculated using mFold [45] were decreased maximally by 60 °C after S-arm truncations (Fig. S4). Little background cleavage in the absence of PLL-g-Dex was observed for the truncated MNAzymes (Fig. 2a). The S/B ratios were increased by the truncation, especially, Mz(6,7) showed S/B 286, while that of long-armed Mz(10,11) was 32.3 (Table 1). As expected, S-arms truncation adversely impacted the desired effector-dependent signal reaction in the absence of PLL-g-Dex. Signal reaction rate was decreased (Fig. 2c and e) because the MNAzyme assembly involving a substrate and an effector was also destabilized by the S-arms truncation. In the presence of PLL-g-Dex, the background cleavage at 30 min decreased from 17% for Mz(10,11) to less than 3% for the truncated MNAzymes (Fig. 2b). Notably, the signal reactions of the MNAzymes with short S-arms were significantly enhanced by PLL-g-Dex (Fig. 2d); reaction rates were increased up to 210 folds higher than that of longarmed Mz(10,11) in the absence of PLL-g-Dex. The S/B ratios of Mz (7,8), Mz(6,7), and Mz(5,6) were increased to 1.47 × 103, 3.08 × 103, and 1.13 × 103, respectively (Table 1). As the S-arm was truncated, the optimal temperatures were decreased; however, reaction rates remained high in the presence of PLL-g-Dex (Fig. 2f). The effects of PLL-g-Dex and S-arm truncation were synergistic. In comparison with Mz(10,11) without PLL-g-Dex, the addition of PLL-g4

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Fig. 2. Substrate cleavage as a function of time and reaction rate as a function of temperature for MNAzymes with different S-arm lengths indicated by color. Percent cleavage without miR-21 effector in (a) the absence and (b) the presence of PLL-g-Dex. Percent cleavage in the presence of 20 nM miR-21 effector in (c) the absence and (d) the presence of PLL-g-Dex. Reaction rate versus temperature for effector-dependent reaction in (e) the absence and (f) the presence of PLL-g-Dex. Experimental conditions: 50 mM HEPES, 150 mM NaCl, pH 7.3, 5 mM MnCl2, 100 nM substrate, 20 nM enzyme, and 0 or 20 nM miR-21 effector without or with PLL-g-Dex (N/P of 2). Reactions were performed at the optimal temperatures for each MNAzyme (Table 1). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

reduces substrate affinity for the MNAzyme. Even though effector-induced destabilization occurs, the reaction rate of the Mz(6,7)-catalyzed effector dependent reaction (9.42 × 10−1 min−1) was higher than that of the long-armed MNAzyme (Mz(10,11), 3.91 × 10−1 min−1) in the presence of PLL-g-Dex indicating sufficient substrate assembly.

MNAzymes having short S-arms were limited by insufficient k1. To test this assumption, substrate concentration dependence was assessed. The twice higher reaction rate of Mz(6,7) and Mz(5,6) but not that of Mz (10,11) was observed when substrate concentration was increased 2 folds (Fig. S5), confirming the rate-limiting step was changed. Under this condition, Mz(6,7) exhibited the highest activity among MNAzymes examined (Fig. S5). Unexpectedly, in the presence of PLL-g-Dex, a lower Tm was observed in the presence of the effector than in its absence for the Mz(6,7) (Fig. 3f, compare red solid and red dashed lines). The Tm of Mz(6,7) was approximately 10°C lower in the presence of effector suggested that the effector binding to MNAzymes destabilized the four-stranded mature complex. This effector-induced destabilization was also observed for other short-armed MNAzyme constructs of different sequences (Fig. S6). The reason for this effect is still unclear. We speculate that the effector binding alters the MNAzyme conformation and allosterically

3.4. Effector sensitivity To further evaluate the analytical performance of the MNAzyme system, we analyzed the concentration-dependent responses to the miR21 effector of Mz(10,11) and Mz(7,8). Without PLL-g-Dex, substrate cleavage increased with the increased effector concentration, but only about 5% cleavage was observed at 10 min with 20 nM of effector for both Mz(10,11) (Fig. 4a) and Mz(7,8) (Fig. 4b). The limit of detection (LoD) was calculated based on the recommendations of IUPAC [46] and ACS [47], which equal to 3σ/slope of a calibration curve, where σ is the 5

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Fig. 3. Thermal denaturation analysis. Sequences and locations of fluorophore and quencher on (a) Mz(10,11) and (b) Mz(6,7). Thermal dissociation curves of (c) Mz(10,11) without PLL-g-Dex, (d) Mz (10,11) with PLL-g-Dex, (e) Mz(6,7) without PLL-gDex, and (f) Mz(6,7) with PLL-g-Dex. Black lines indicate the fluorescence intensity of the free substrate. Red solid and red dashed lines represent the fluorescence intensity of condition in the presence or absence of effector, respectively. Experimental conditions: 50 mM HEPES, 150 mM NaCl, pH 7.3, 5 mM MnCl2, 50 nM all-DNA substrate, 0 or 50 nM enzyme, and 0 or 50 nM effector without or with PLL-g-Dex (N/P of 2). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

The MNAzyme reaction is isothermal, thus the signal is measured at a constant temperature so a thermocycler is not required. The simple, protein-free system can be operated under mild conditions in one pot and requires no labor-intensive steps. Results can be obtained in less than 1 h, whereas PCR-based detection methods require more than 3 h. Our MNAzyme-based method has comparable sensitivity and several advantages compared to common isothermal amplification methods, such as rolling circle amplification [49–51], catalytic hairpin assembly [52–55], and hybridization chain reaction [56–58]. PLL-g-Dex permits rapid and sensitive miRNA detection without complicated signal amplification procedures. The MNAzyme measurement can be performed by non-experts and does not require sophisticated instruments. In addition, MNAzymes can be tailored to detect various target sequences without need for complicated primer and probe design strategies.

standard deviation of the blank. The LoD without PLL-g-Dex for Mz (10,11) and Mz(7,8) were 2.0 × 10−10 M, and 9.0 × 10−11 M, respectively. In contrast, in the presence of PLL-g-Dex, the effector was detected at picomolar concentrations with both MNAzymes. The LoD values of Mz(10,11) and Mz(7,8) were 1.0 × 10−11 M and 3.8 × 10−13 M, respectively. The calibration curve of the percent cleavage induced by Mz(7,8) in the presence of PLL-g-Dex had a good linear correlation with the miR-21 concentration (R2 = 0.998), showing the reproducibility of our method (Fig. S7). The truncated MMAzyme Mz(7,8) with PLL-g-Dex was 500-fold more sensitive than the long-armed Mz(10,11) without PLL-g-Dex. The plots of percent substrate cleavage at 30 min and the logarithm of the effector concentration clearly demonstrate the improvement in sensitivity in the presence of PLL-g-Dex (Fig. 4c and d). To evaluate the applicability of the MNAzyme system in real biological samples, MNAzyme activity was analyzed in the presence of total RNA extracted from HeLa cells. MNAzyme activity increased as expected with the amount of synthetic miRNA target added to the reaction solution (Fig. S8). The determined sensitivity of MNAzyme in the presence of the copolymer is comparable to that of commercial miRNA detection kits based on qRT-PCR such as TaqMan Advanced miRNA Assays (ThermoFisher) and miRCURY LNA miRNA PCR Assays (QIAGEN) [48].

3.5. Selectivity in the multiplex format Multiplexed assays are necessary for accurate and efficient disease diagnosis [59]. We designed dual-MNAzyme assays using two MNAzymes to detect miRNAs from the miR-21 family, miR-21 and miR-21b (Fig. 5a and b); and miRNAs from the let-7 family, let-7a and let-7d 6

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Fig. 4. Effector concentration dependency. Percent substrate cleavage as a function of time in the presence of indicated concentrations of effector miR-21 by (a) Mz(10,11) and (b) Mz(7,8); and cleavage at 30 min versus the logarithm of miR-21 concentration for (c) Mz(10,11) and (d) Mz(7,8). Experimental conditions: 50 mM HEPES, 150 mM NaCl, pH 7.3, 5 mM MnCl2, 100 nM substrate, 20 nM enzyme, and 0–20 nM miR-21 effector without or with PLL-g-Dex (N/P of 2). Reaction temperatures were at the optimal temperatures for each MNAzyme (Table 1).

designed for multiplexed assays were enhanced in the presence of PLLg-Dex regardless of substrate and target sequences. Results confirmed the generality of the enhancement by PLL-g-Dex. The abilities of the MNAzymes to discriminate between targets of high sequence similarity were examined. All MNAzymes produced high signal in the presence of fully matched targets (Fig. 5e–h). For the MNAzyme designed for detection of miR-21 family, the signal in the presence of an oligonucleotide with five mismatches was close to the background level. In the miR-21 assay, no signal was observed in the presence of miR-21b (Fig. 5e). Complete discrimination was also observed in the miR-21b assay (Fig. 5f). For the MNAzyme designed for detection of let-7 family miRNAs, a moderate response was observed in the presence of the mismatched targets in the let-7a and let-7d assays (Fig. 5g and h). This was not unexpected as the let-7 miRNAs have only two base differences. Discrimination of miRNA sequences differing by a single nucleotide was demonstrated (Fig. S10). Assuming 100% efficiency for the reaction rate in the presence of the perfect match target let-7a, only 7.1% and 5.7% efficiency due to cross-hybridization

(Fig. 5c and d). As each dual assay was designed to detect targets with high sequence homology, this experimental design allowed us to examine the selectivity of the system. Two substrates of dual assay have different sequences and different fluorophore-quencher pairs. This allowed the activity of two MNAzymes to be monitored at different excitation (λex) and emission (λem) wavelengths. The cleavage of the substrate labeled with FITC and BHQ-1 was detected at λex 494 nm and λem 520 nm, whereas that of the substrate labeled with TAMRA and BHQ-2 was detected at λex 556 nm and λem 580 nm. Similar optimal temperature of two MNAzymes within each dual assay was achieved by tuning S-arms lengths. First, reactivity and selectivity of each MNAzyme were separately evaluated. For all MNAzymes, PLL-g-Dex increased the optimal reaction temperature approximately 10 °C and accelerated target-dependent reaction rates by two orders of magnitude (Fig. S9 and Table S1). Selectivity was enhanced by the copolymer; S/B was up to 60-fold higher at 20 nM target concentrations in the presence compared to the absence of PLL-g-Dex (Table S1). The activity and selectivity of MNAzymes 7

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Fig. 5. MNAzyme for dual targets detection. Sequences of the MNAzymes recognizing targets (a) miR-21, (b) miR-21b, (c) let-7a and (d) let-7d. Selectivity of (e) Mz(8,8)-miR-21 (λex = 494 nm (f) Mz(9,8)-miR-21b and λem = 520 nm), (λex = 556 nm and λem = 580 nm), (g) Mz(8,8)-let7a (λex = 494 nm and λem = 520 nm), and (h) Mz (9,8)-let-7d (λex = 556 nm and λem = 580 nm). Experimental conditions: 50 mM HEPES, 150 mM NaCl, pH 7.3, 5 mM MnCl2, 100 nM substrate, 20 nM enzyme, 0 or 20 nM target (fully matched or mismatched), and PLL-g-Dex N/P of 2. Reaction temperature 30 °C for miR-21 and miR-21b assay and 37 °C for let-7 and let-7d assay.

8

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Fig. 6. Selectivity of dual-MNAzyme assays. Activities of (a) miR-21/miR21b and (b) let-7a/let-7d dual assays compared to single assays with targets present at 20 nM (fully matched or mismatched). Responses of dualMNAzyme assay for (c) miR-21/miR21b and (d) let-7a/let-7d when fully matched and mismatched targets are present at different ratios. Substrate cleavage of Mz(8,8)-miR-21 and Mz (8,8)-let-7a was detected at and λem = 520 nm. λex = 494 nm Substrate cleavage of Mz(9,8)-miR-21b and Mz(9,8)-let-7d was detected at and λem = 580 nm. λex = 556 nm Experimental conditions: 50 mM HEPES, 150 mM NaCl, pH 7.3, 5 mM MnCl2, 100 nM each substrate, 20 nM each enzyme, 0–20 nM target as indicated, and PLL-g-Dex (N/P of 2). Reaction temperatures were 30 °C and 37 °C for miR-21/miR-21b and let-7a/ let-7d assays, respectively.

(Fig. 7b, black line). In contrast, when miR-21 was incubated with RNase A in the presence of PLL-g-Dex, MNAzyme activity (Fig. 7b, green line) was comparable to the condition without RNase A (Fig. 7b, blue line). This indicated that the copolymer effectively prevents target miR-21 degradation by RNase A. Furthermore, we also observed target concentration dependency when miR-21 was incubated with RNase A in the presence of PLL-g-Dex (Fig. 7c). Results clearly demonstrated the protective effect of PLL-g-Dex against RNase A degradation.

occurred in the presence of let-7d, which has two base differences, and let-7f, with a single mismatch, respectively. To verify the feasibility of dual-MNAzyme assay, the two MNAzymes designed to recognize miR-21 and miR-21b and their substrates were combined within the same reaction solution. Substrate cleavages were monitored in the presence of either miR-21 or miR-21b targets. The selectivity of the miR-21/miR-21b dual assay was comparable to the single assay (Fig. 6a). Similar results were obtained by analysis of data from the let-7a/let-7d dual assay (Fig. 6b). Interestingly, the false-positive signal was lower in the dual assay of the MNAzymes targeting let-7a and let-7d compared to those in the single assays. In the presence of two MNAzymes, competitive binding to fully matched targets presumably decreased the availability of mismatched target and consequently minimized false-positive signal (Fig. 6b). Furthermore, when two targets were presence at different ratios, signals in the dual assays correlated with specific target concentrations (Fig. 6c and d).

4. Discussion In general, the counterion condensation that occurs in the local environment of nucleic acids partially neutralizes anionic charges and stabilizes the nucleic acid complexes. Counterion condensation contributes to the entropic barrier to oligonucleotide hybridization. We previously reported that PLL-g-Dex reduces electrostatic repulsion between the negative charges on the polynucleotide backbone and eliminates the counterion condensation effect [25]. When the copolymer interacts with a nucleic acid strand, counterions are released into the bulk solution which increases the entropy of the system and contributes to the enhanced thermal stability of nucleic acid structure. In the presence of PLL-g-Dex, melting temperatures and hybridization rates of DNA duplexes and triplexes are significantly increased [24–28]. In this study, the thermal stability of an MNAzyme was enhanced by the copolymer (Fig. 3), which correlated with an increased optimal temperature and reaction rate (Fig. 2 and Table 1). The copolymer enhances catalytic activity of MNAzyme by two mechanisms: It facilitates multi-strand hybridization and stabilizes the MNAzyme-substrate-effector complex. It was reported that the substrate cleavage rate of a DNAzyme is limited by the hybridization rate of the substrate with the DNAzyme [62]. Hybridization of substrate and effector with two partzymes is considered to be the rate-limiting step of MNAzyme catalysis. MNAzyme activity is compromised by insufficient hybridization stability, and this effect is more pronounced with short

3.6. Protecting from RNase degradation with PLL-g-Dex In practical situations, MNAzyme reactions could be impeded by contamination of proteinous ribonucleases (RNases). Especially, RNases are ubiquitous and can cause RNA degradation and disorder MNAzyme reactions. We previously reported that the cationic copolymer protected siRNA from nucleases in serum and extended its blood circulation time in mouse [60,61]. We hypothesized that the PLL-g-Dex could prevent miRNA target degradation and improve target selectivity in the presence of RNase A. The miR-21 was firstly incubated with RNase A in the presence or absence of PLL-g-Dex. Then MNAzyme activity was evaluated at similar PLL-g-Dex concentration (summary of experimental conditions was shown in Fig. 7a). When miR-21 was incubated with RNase A in the absence of PLL-g-Dex, low MNAzyme activity was observed due to miR-21 degradation by RNase A (Fig. 7b, red line). The activity was almost similar to the reaction in the absence of miR-21 9

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Fig. 7. MNAzyme activities in the presence of RNase A. (a) Summary of reaction conditions and legend color indicated in (b) and (c). (b) Percent of substrate cleaved by MNAzyme under different conditions. (c) Reaction rate constant in the presence of 20 nM miR-21 (solid fill) or 100 nM miR-21 (stripes). Experimental condition 1–4 contained 100 nM substrate and 20 nM partzymes in 50 mM HEPES, 150 mM NaCl, 5 mM MnCl2 (pH 7.3). The N/P ratio during MNAzyme reaction was kept as 5 for all conditions, the copolymer was added before incubated with 100 ng/mL RNase for conditions 1, 3, and 4, and added after incubation with RNase for condition 2. Condition 1 was a negative control without miR-21 and RNase A and condition 4 was a positive control that contained miR-21 in the absence of RNase A. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

However, in the absence PLL-g-Dex, S-arms truncation also destabilized MNAzyme complex involving a substrate and an effector, resulting in loss of signal efficiency and impeded the improvement of S/B ratio. Substantial reduction in signal reaction rate of the short-armed MNAzyme was correlated with decreased thermal stability (Fig. 3 and Table 1). Previous studies investigated a relationship between catalytic efficiency and S-arms length demonstrated that catalytic efficiency of DNAzyme having too short S-arms was limited by poor substrate association rate [62,66]. Combination of PLL-g-Dex and S-arms truncation solved the drawbacks of individual strategy. The combination synergistically improved catalytic efficiency and selectivity of MNAzymes, which led to a significant increase of S/B ratio compare to individual strategy. Sufficient substrate binding rate, k1, in the presence of PLL-g-Dex allowed background to be suppressed by S-arms truncation without causing adverse effect to signal reaction. In addition, signal reaction rate of short-armed MNAzymes Mz(7,8) and Mz(6,7) in the presence of PLL-g-Dex was increased even higher than the unmodified MNAzyme Mz(10,11). An increased signal reaction rate by S-arms truncation reflects a change in the rate-limiting step of Mz(10,11) from k1 to k3 in the presence of PLLg-Dex. Two times higher substrate concentration did not increase reaction rate of Mz(10,11) confirmed that its catalytic efficiency in the presence of PLL-g-Dex was not limit by k1 (Fig. S5). Instead, S-arms truncation increased reaction rate as k3 was increased. When S-arms were truncated shorter than an optimal length, catalytic efficiency of Mz(5,6) was decreased because of insufficient k1. Reaction rates of short-armed MNAzymes were increased with increased substrate concentration as k1 was promoted (Fig. S5). These results suggested that catalytic efficiency of MNAzyme was improved when k1 and k3 were optimally tuned. In the case of DNAzyme, more than 20-fold increased activity was obtained by tuning number of modified nucleotides to increase k1 and S-arms length to increase k3 [19]. In the present study, PLL-g-Dex and S-arms truncation improved activity maximally 210-fold (Table 1). The effect of DNAzyme S-arms length reduction varied with

effector strands. In the presence of the copolymer, an assembly with short effector miR-21 (22 nt) and turnover reactions were successfully improved which increased reaction rate under multiple turnover condition as high as that of MNAzyme having long effector (29 nt) under single turnover reaction. PLL-g-Dex accelerated MNAzyme turnover by increase k1 and stabilize MNAzyme complex (increase k1/k-1). Previously, nucleotide modifications are used to improve helical thermostability of nucleic acids duplex, such as 2′-O-methylated nucleotides [63] and locked nucleic acid [64,65]. Catalytic activity of DNAzyme enhanced by substitution of modified nucleotides into S-arms were previously reported [19–21]. However, excessive modifications can interfere with the correct folding of the catalytic core. Also, it can prevent product release and decreased substrate turnover [19,62]. In contrast, the stabilization effect of PLL-g-Dex attributed to increasing in the on-rate of DNA duplex formation rather than decrease in dissociation rate. According to this, the addition of PLL-g-Dex promotes MNAzyme association k1 while not preventing release of cleaved substrate k3. We previously reported significant enhanced catalytic efficiency of DNAzyme [39] and MNAzyme [23] by PLL-g-Dex even under multiple turnover condition. Reaction rate of MNAzyme was significantly increased by two orders of magnitude. Similar degree of activity enhancement by PLL-g-Dex was achieved in the present study, even shorter effector was used. Effector-dependent signal reaction rate of Mz (10,11) increased by 79-times in the presence of PLL-g-Dex (Table 1). However, the improvement of S/B ratio was compromised by the effector-independent background reaction (Scheme 1b). Therefore, additional strategy was combined to improve MNAzyme selectivity. The effector-independent background reaction was minimized by Sarms truncation. According to Fig. S3, binding stability between partzymes and substrate influences background cleavage reaction. Therefore, S-arms were shortened to destabilize enzyme-substrate complex in the absence of the effector (or decrease k’1/k’-1). We obtained lower background reaction rate for the truncated MNAzymes Mz(7,8), Mz (6,7), and Mz(5,6) compare to the unmodified MNAzyme Mz(11,10). 10

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substrate sequence and asymmetric truncation of each arm [18]. Therefore, kinetic tuning has to be studied systematically and it might not be optimal for different MNAzyme sequences.

[9]

5. Conclusion

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We demonstrated that the cationic copolymer PLL-g-Dex promoted the assembly of multi-stranded MNAzymes and selectively enhanced the effector-dependent signal reaction. For the first time, we described a strategy for tuning MNAzyme kinetics with regard to both catalytic efficiency and selectivity by addition of PLL-g-Dex and S-arm truncation to optimize the association rate k1 and the dissociation rate k3. The activity of the copolymer also allowed S-arm truncation to minimize background and further accelerate enzymatic turnover, whereas S-arm truncation in the absence of PLL-g-Dex results in a considerable loss of the MNAzyme reactivity. The sensitivity of MNAzyme for miRNA detection was improved three orders in the presence of PLL-g-Dex. The MNAzyme reaction can be operated under isothermal and mild conditions without sophisticated instruments. Furthermore, the decrease in optimal reaction temperature due to S-arm truncation resulted in a highly active MNAzyme with an optimum near ambient temperature, which is preferred for applications such as point-of-care diagnosis. Though mechanisms underlying selective enhancement of signal by PLL-g-Dex are not fully understood, the effect was general as shown by analysis of a number of MNAzymes. The synergistic improvements resulting from S-arm truncation and PLL-g-Dex allowed us to design sensitive and selective dual-MNAzyme systems that detect highly similar miRNAs in the same solution. In addition, the copolymer conferred substantial protection from proteinous RNase degradation, which significantly improved MNAzyme activity in the presence of RNase A. The PLL-g-Dex chaperoning DNA-based sensors have a potential for use as simple, rapid, highly sensitive assays for nucleic acid.

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[15]

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[19]

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Conflicts of interest

[21]

The authors declare no conflict of interest. [22]

Acknowledgments This work was financially supported by Center of Innovation (COI) Program (JPMJCE1305), Japan Science and Technology Agency (JST), and by KAKENHI (15H01807) from Japan Society for the Promotion of Science.

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biomaterials.2019.119535.

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