DNA-Based Enzyme Reactors and Systems

This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s): Title: Year: Version: Please cite the original version: All material supplied via JYX is protected by copyright and other intellectual property rights, and duplication or sale of all or part of any of the repository collections is not permitted, except that material may be duplicated by you for your research use or educational purposes in electronic or print form. You must obtain permission for any other use. Electronic or print copies may not be offered, whether for sale or otherwise to anyone who is not an authorised user. DNA-Based Enzyme Reactors and Systems Linko, Veikko; Nummelin, Sami; Aarnos, Laura; Tapio, Kosti; Toppari, Jussi; Kostiainen, Mauri A. Linko, V., Nummelin, S., Aarnos, L., Tapio, K., Toppari, J., & Kostiainen, M. A. (2016). DNA-Based Enzyme Reactors and Systems. Nanomaterials, 6(8), Article 139. https://doi.org/10.3390/nano6080139 2016 nanomaterials Review DNA-Based Enzyme Reactors and Systems Veikko Linko 1,*, Sami Nummelin 1, Laura Aarnos 1, Kosti Tapio 2, J. Jussi Toppari 2 and Mauri A. Kostiainen 1,* 1Biohybrid Materials, Department of Biotechnology and Chemical Technology, Aalto University, P.O. Box 16100, Aalto 00076, Finland; [email protected] (S.N.); [email protected] (L.A.) 2 Department of Physics, University of Jyvaskyla, Nanoscience Center, P.O. Box 35, Jyväskylä 40014, Finland; [email protected] (K.T.); [email protected] (J.J.T.) *Correspondence: [email protected] (V.L.); [email protected] (M.A.K.); Tel.: +358-45-673-9997 (V.L.); +358-50-362-7070 (M.A.K.) Academic Editor: Leonid Gurevich Received: 8 June 2016; Accepted: 19 July 2016; Published: 27 July 2016 Abstract: During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme) cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications. Keywords: DNA nanotechnology; DNA origami; self-assembly; enzyme; cascade reactions; DNA nanodevice; DNA sensors; drug-delivery; nanomedicine 1. Introduction In order to maintain complex metabolic pathways, nature uses compartmentalization and spatial organization of metabolically active units to separate specialized functions, control activity and gain specificity. In the cell, specific organelles control the location and crowding of enzymes, which has a profound effect on their spatial action, and ultimately allows different metabolic pathways to operate at the same time in close proximity, but in different compartments. For example, electron transport and oxidative phosphorylation are handled by the mitochondrion, whereas at the same time glycolysis and fatty acid biosynthesis take place in the cytosol. Furthermore, multiple enzymes responsible for the individual reaction steps in a metabolic pathway are often combined into a single multifunctional enzyme or complex in order to enhance and control the reaction cascade or cycle. Fatty acid synthase is one of the prime examples: in animals it combines two identical protein chains that contain seven different catalytic activities required for the biosynthesis of fatty acids. Although highly desirable, the programming of chosen reaction cascades and creating artificial systems that can position and confine different enzymes is still far from the complexity achieved by nature. Controlling chemical reactions by using self-assembled nanoscale reactors has consequently emerged as an active area of research [1]. Simple compartmentalization of enzymes has already been achieved by using different nanoscale reactors. Examples of such systems include sol-gel materials [ 2 ], polymersomes [ 3 ], protein cages [ 4 – 6 ] and crystalline structures [ 7 – 9 ]. Porous polymersomes were used as nanoreactors to anchor three different enzymes into separate locations: the lumen, bilayer membrane and surface. Protein cages, such as virus-like particles, have been utilized to pack different Nanomaterials 2016,6, 139; doi:10.3390/nano6080139 www.mdpi.com/journal/nanomaterials Nanomaterials 2016,6, 139 2 of 16 enzymes that perform a coupled cascade reaction densely inside a porous protein shell. Finally, for example, metal-organic frameworks have been designed to trap and encapsulate enzymes and shown to prevent their aggregation and denaturation. All of the above-mentioned systems are prime examples on how the positioning, separation and clustering of enzymes can be controlled. In this review, we focus on complexes and systems that involve functional enzymes and novel DNA nanostructures (an example of such a system is depicted in Figure 1). These sophisticated DNA nanostructures can be programmed to form precise and controllable arrangements of enzymes at the nanoscale, and these systems are particularly engaging for various applications in bioengineering and nanomedicine. We have divided this review into four main sections. First, we briefly summarize the development in structural DNA nanotechnology and discuss how DNA motifs can be combined with functional enzymes (Section 2). Section 3is devoted to (static) enzymatic nanoreactors, and Section 4 covers the enzymatic regulatory devices with mechanical function. Finally, in Section 5, containers and carriers for protecting and delivering enzymes are discussed. Nanomaterials 2016, 6, 139 2 of 16 different enzymes that perform a coupled cascade reaction densely inside a porous protein shell. Finally, for example, metal-organic frameworks have been designed to trap and encapsulate enzymes and shown to prevent their aggregation and denaturation. All of the above-mentioned systems are prime examples on how the positioning, separation and clustering of enzymes can be controlled. In this review, we focus on complexes and systems that involve functional enzymes and novel DNA nanostructures (an example of such a system is depicted in Figure 1). These sophisticated DNA nanostructures can be programmed to form precise and controllable arrangements of enzymes at the nanoscale, and these systems are particularly engaging for various applications in bioengineering and nanomedicine. We have divided this review into four main sections. First, we briefly summarize the development in structural DNA nanotechnology and discuss how DNA motifs can be combined with functional enzymes (Section 2). Section 3 is devoted to (static) enzymatic nanoreactors, and Section 4 covers the enzymatic regulatory devices with mechanical function. Finally, in Section 5, containers and carriers for protecting and delivering enzymes are discussed. Figure 1. A schematic view of an enzymatic nanoreactor built from DNA ([S] = substrate, [P] = product). By taking advantage of the high addressability and modularity of the DNA nanostructures, enzymes can be attached and arranged with nanometer-scale precision. As an example, glucose oxidase (GOx, purple)–horseradish peroxidase (HRP, green) cascade pairs have been assembled into a confined reaction space provided by two tubular DNA origami nanostructures (orange and yellow cages). 2. Building with DNA Molecules and Enzymes 2.1. DNA Nanostructures Nadrian ‘Ned’ Seeman postulated around 30 years ago that deoxyribose (DNA) molecules could be used as building material in creating complex predesigned nanostructures through molecular selfassembly [10]; sequence-complementary parts of single-stranded DNA (ssDNA) molecules can be hybridized into double-stranded DNA (dsDNA) domains (via Watson-Crick base-pairing) and therefore into larger programmed shapes. Since then, structural DNA nanotechnology has enjoyed a rapid progress; numerous complex nanostructures and different fabrication techniques have been introduced [11] (Figure 2). A great deal of the first compelling DNA assemblies were based on tilelike structures that enabled fabrication of two-dimensional (2D) [12] and three-dimensional (3D) crystals [13], but nevertheless, the huge upturn in the field was the invention of the ‘DNA origami’ technique [14] (Figure 2a). The origami approach is based on folding a long single-stranded DNA scaffold strand into a desired shape with the help of a set of short oligonucleotides (staples), and it has now become a widely accessible and exploited method to fabricate custom, modular and spatiallywell-defined 2D [14] and 3D nanostructures with complex curvatures, bends and twists [15–18] (see Figure 2b,c). Later on, methods based on scaffold-free fabrication [19] (Figure 2d), polyhedral rendering [20,21] (Figure 2e) and shape-complementarity [22] (Figure 2f) were introduced. Figure 1. A schematic view of an enzymatic nanoreactor built from DNA ([S] = substrate, [P] = product). By taking advantage of the high addressability and modularity of the DNA nanostructures, enzymes can be attached and arranged with nanometer-scale precision. As an example, glucose oxidase (GOx, purple)–horseradish peroxidase (HRP, green) cascade pairs have been assembled into a confined reaction space provided by two tubular DNA origami nanostructures (orange and yellow cages). 2. Building with DNA Molecules and Enzymes 2.1. DNA Nanostructures Nadrian ‘Ned’ Seeman postulated around 30 years ago that deoxyribose (DNA) molecules could be used as building material in creating complex predesigned nanostructures through molecular self-assembly [ 10 ]; sequence-complementary parts of single-stranded DNA (ssDNA) molecules can be hybridized into double-stranded DNA (dsDNA) domains (via Watson-Crick base-pairing) and therefore into larger programmed shapes. Since then, structural DNA nanotechnology has enjoyed a rapid progress; numerous complex nanostructures and different fabrication techniques have been introduced [ 11 ] (Figure 2). A great deal of the first compelling DNA assemblies were based on tile-like structures that enabled fabrication of two-dimensional (2D) [ 12 ] and three-dimensional (3D) crystals [ 13 ], but nevertheless, the huge upturn in the field was the invention of the ‘DNA origami’ technique [ 14 ] (Figure 2a). The origami approach is based on folding a long single-stranded DNA scaffold strand into a desired shape with the help of a set of short oligonucleotides (staples), and it has now become a widely accessible and exploited method to fabricate custom, modular and spatially-well-defined 2D [ 14 ] and 3D nanostructures with complex curvatures, bends and twists [15–18] (see Figure 2b,c). Later on, methods based on scaffold-free fabrication [ 19 ] (Figure 2d), polyhedral rendering [ 20 , 21 ] (Figure 2e) and shape-complementarity [ 22 ] (Figure 2f) were introduced. Nanomaterials 2016,6, 139 3 of 16 Nanomaterials 2016, 6, 139 3 of 16 Figure 2. (a) A DNA origami technique. A long scaffold strand is folded into a desired shape with the help of short staple strands [14]; (b) Multilayer DNA origami in square and honeycomb lattice [15,16]; (c) DNA origami with curvatures and bends [17,18]; (d) Scaffold-free fabrication of DNA nanoshapes. Numerous target shapes can be fabricated by selecting subsets of strands from the cubic-like ‘molecular canvas’ [19]; (e) A fully automated top-down design method to create meshed DNA origami structures [21]; (f) DNA origami structures can be glued together by taking advantage of the blunt-end stacking and the shape-complementarity of the origami units [22]. (a) is reproduced with permission from [14]. Copyright Nature Publishing Group, 2006. (b) is reproduced with permission from [16]. Copyright Nature Publishing Group, 2011. A sphere in (c) is reproduced with permission from [17]. Copyright The American Association for the Advancement of Science, 2011. A gear-like object in (c) is reproduced with permission from [18]. Copyright The American Association for the Advancement of Science, 2009. (d) is reproduced with permission from [19]. Copyright The American Association for the Advancement of Science, 2012. (e) is reproduced with permission from [21]. Copyright The American Association for the Advancement of Science, 2016. (f) is reproduced with permission from [22]. Copyright The American Association for the Advancement of Science, 2015. In general, the DNA-based assembly of nanostructures is a highly parallel technique, and the nanometer-scale addressability of the created objects makes it an intriguing approach for developing novel bionanotechnological applications [11]. To date, loads of implementations based on DNA nanostructures have been presented, such as tunable plasmonic devices and metallic nanoshapes [23,24], rulers for optical imaging [25], structures for nanoelectronics [26,27], artificial ion channels for transporting or sequencing molecules [28], and nanorobots for targeted drug delivery [29]. Moreover, as discussed in this review, DNA nanostructures provide an excellent foundation for designing enzymatic reactors and complex catalytic systems at the nanoscale. 2.2. DNA-Enzyme Conjugates and Arrays As discussed above, DNA structures can be used as templates for various molecules, inorganic nanoparticles, and equally for functional enzymes [30]. Enzymes can be conjugated directly to an oligonucleotide (part of a DNA structure) or they can be attached to DNA through a specific binding motif [30]. In general, it is important that the enzyme activity is retained in the conjugation; a chosen enzyme should not be modified chemically or genetically [31]. For example, sequence-specific DNAbinding proteins can be used as adaptors in attachment [32], and their use can help to maintain the enzyme activity in the conjugation. To date, there exist numerous reports of utilizing simple nucleic acid motifs to assemble functional enzymes and to organize chemical reactions with programmability [33–36]. In addition, it has been shown that by utilizing DNA-based self-assembly, structurally-well-defined protein arrays [30,37] and DNA-enzyme crystals [7] can be created. Along these lines, this review discusses recent progress in creating smart enzyme reactors, dynamic regulators, protein containers and carriers by taking advantage of state-of-the-art DNA nanostructures, such as DNA origami. 3. Enzyme Reactors and Cascades Figure 2. ( a ) A DNA origami technique. A long scaffold strand is folded into a desired shape with the help of short staple strands [ 14 ]; ( b ) Multilayer DNA origami in square and honeycomb lattice [ 15 , 16 ]; ( c ) DNA origami with curvatures and bends [ 17 , 18 ]; ( d ) Scaffold-free fabrication of DNA nanoshapes. Numerous target shapes can be fabricated by selecting subsets of strands from the cubic-like ‘molecular canvas’ [ 19 ]; ( e ) A fully automated top-down design method to create meshed DNA origami structures [ 21 ]; ( f ) DNA origami structures can be glued together by taking advantage of the blunt-end stacking and the shape-complementarity of the origami units [ 22 ]. ( a ) is reproduced with permission from [ 14 ]. Copyright Nature Publishing Group, 2006. ( b ) is reproduced with permission from [ 16 ]. Copyright Nature Publishing Group, 2011. A sphere in ( c ) is reproduced with permission from [ 17 ]. Copyright The American Association for the Advancement of Science, 2011. A gear-like object in ( c ) is reproduced with permission from [ 18 ]. Copyright The American Association for the Advancement of Science, 2009. ( d ) is reproduced with permission from [ 19 ]. Copyright The American Association for the Advancement of Science, 2012. ( e ) is reproduced with permission from [ 21 ]. Copyright The American Association for the Advancement of Science, 2016. ( f ) is reproduced with permission from [22]. Copyright The American Association for the Advancement of Science, 2015. In general, the DNA-based assembly of nanostructures is a highly parallel technique, and the nanometer-scale addressability of the created objects makes it an intriguing approach for developing novel bionanotechnological applications [ 11 ]. To date, loads of implementations based on DNA nanostructures have been presented, such as tunable plasmonic devices and metallic nanoshapes [23,24] , rulers for optical imaging [ 25 ], structures for nanoelectronics [ 26 , 27 ], artificial ion channels for transporting or sequencing molecules [ 28 ], and nanorobots for targeted drug delivery [ 29 ]. Moreover, as discussed in this review, DNA nanostructures provide an excellent foundation for designing enzymatic reactors and complex catalytic systems at the nanoscale. 2.2. DNA-Enzyme Conjugates and Arrays As discussed above, DNA structures can be used as templates for various molecules, inorganic nanoparticles, and equally for functional enzymes [ 30 ]. Enzymes can be conjugated directly to an oligonucleotide (part of a DNA structure) or they can be attached to DNA through a specific binding motif [ 30 ]. In general, it is important that the enzyme activity is retained in the conjugation; a chosen enzyme should not be modified chemically or genetically [ 31 ]. For example, sequence-specific DNA-binding proteins can be used as adaptors in attachment [ 32 ], and their use can help to maintain the enzyme activity in the conjugation. To date, there exist numerous reports of utilizing simple nucleic acid motifs to assemble functional enzymes and to organize chemical reactions with programmability [ 33 – 36 ]. In addition, it has been shown that by utilizing DNA-based self-assembly, structurally-well-defined protein arrays [ 30 , 37 ] and DNA-enzyme crystals [ 7 ] can be created. Along these lines, this review discusses recent progress in creating smart enzyme reactors, dynamic regulators, protein containers and carriers by taking advantage of state-of-the-art DNA nanostructures, such as DNA origami. Nanomaterials 2016,6, 139 4 of 16 3. Enzyme Reactors and Cascades An enzyme reactor typically contains one or more enzymes, which catalyze a desired reaction. The purpose of the enzyme reactor is usually to maximize the reaction efficiency via compartmentalization or by bringing the reaction counterparts in close proximity to each other. By utilizing designed DNA nanostructures with high addressability, enzymes can be attached to them with nanoscale precision. This is a key factor for enzyme functions; a substrate can only bind to an enzyme in a specific orientation, and on the other hand, the proximity of the compounds provided by the DNA templates could significantly enhance the enzymatic reaction rates [ 38 , 39 ]. In addition, it is essential to control the channeling of the substrate and the reaction intermediates of the enzyme cascades [ 40 ]. In many cases, compartmentalization could be used to efficiently separate and arrange simultaneous reactions and reaction compounds similar to complex natural systems [ 41 ]. Moreover, enzyme reactors can be equally utilized to study enzyme functions and reaction pathways [ 42 ]. In this section, recent examples of using DNA nanostructures to build (static) nanoreactors for biosensing and molecular-scale diagnostics are discussed (see also Table 1). Table 1. Examples of DNA-based enzyme reactors and cascades. Type Function Key Aspects A glucose oxidase (GOx) – horseradish peroxidase (HRP) cascade on a DNA origami [43]. The enzyme positions on the DNA origami template can be tuned. The cascade activity is highly dependent on the spacing between the enzymes; the highest activity was found at a 10 nm distance. A GOx-HRP cascade on a DNA origami that can be rolled into tubular shape [44]. The idea is similar to the above, but here the semi-confined tubular geometry could enable shielding. The enzymes in the semi-confined geometry show higher enzymatic activity than the free enzyme controls. A swinging arm between malate dehydrogenase (MDH) and glucose-6-phosphate dehydrogenase (G6pDH) assembled on a double-crossover (DX) DNA tile [45]. The DNA strand acts as a flexible arm that channels the cofactor transfer between the hydrogenases in the complex. The enzyme activity achieved by the swinging arm is significantly higher than in the case of freely diffusing cofactor. A tubular DNA origami nanoreactor with GOx-HRP pairs [46]. The nanoreactor is comprised of two units: GOxand HRP-loaded DNA origamis that can be combined into a complete cascade reactor. Single origami units and the complete reactor equipped with binding sites show higher activity than the controls without binding sites. A xylose reductase (XR) – xylitol dehydrogenase (XDR) cascade on a DNA origami [47]. The enzymes are attached to origami via DNA-binding protein adaptors resulting in an artificial enzyme cascade. The efficiency of the cascade reaction is more dependent on the interenzyme distance than that of the cascade reaction with unimolecular transport between two enzymes. A three-enzyme pathway assembled by a DNA nanostructure [48]. MDH, oxaloacetate decarboxylase (OAD) and lactate dehydrogenase (LDH) are organized at the corners of the triangular DNA nanostructure, thus forming a three-enzyme cascade. Activity of the cascade depends more on the geometric patterns of enzymes than the interenzyme spacings. In the enzyme cascade system presented in Figure 3a, glucose oxidase (GOx) catalyzes the oxidization of glucose (substrate) in the presence of oxygen to generate gluconic acid and a hydrogen peroxide (H2O2) intermediate, which, in turn, serves as a substrate for horseradish peroxidase (HRP) (HRP reduces H 2 O 2 into water). Simultaneously, the presence of H 2 O 2 results in the protonation of the ABTS 2´ (2,2 1 -azinobis-(3-ethylbenzthiazoline-6-sulfonate) dianion, and hence, an ABTS ´ radical anion is generated (ABTS ´ acts as a reporter of the enzyme activity). The diffusion distance of the hydrogen peroxide limits the rate of this enzyme cascade reaction since HRP has a much higher turnover rate than GOx. Fu et al. studied interenzyme substrate diffusion by using a rectangular DNA origami tile as a platform to preorganize GOx-HRP pairs in a distance-dependent manner [ 43 ]. The highest cascade activity was obtained when the interenzyme distance was 10 nm. Importantly, the activity was about 15 times higher than the control sample that contained unbound enzymes. A drastic decrease in activity was observed as the interenzyme distance was adjusted to 20 nm, and the activity was further decreased gradually as the distance was increased up to 65 nm. Nanomaterials 2016,6, 139 5 of 16 Nanomaterials 2016, 6, 139 5 of 16 anion is generated (ABTS− acts as a reporter of the enzyme activity). The diffusion distance of the hydrogen peroxide limits the rate of this enzyme cascade reaction since HRP has a much higher turnover rate than GOx. Fu et al. studied interenzyme substrate diffusion by using a rectangular DNA origami tile as a platform to preorganize GOx-HRP pairs in a distance-dependent manner [43]. The highest cascade activity was obtained when the interenzyme distance was 10 nm. Importantly, the activity was about 15 times higher than the control sample that contained unbound enzymes. A drastic decrease in activity was observed as the interenzyme distance was adjusted to 20 nm, and the activity was further decreased gradually as the distance was increased up to 65 nm. Figure 3. (a) A glucose oxidase (GOx) – horseradish peroxidase (HRP) enzyme cascade pair assembled on a rectangular DNA origami [43]; (b) A rectangular DNA origami shape with attached enzyme cascade pairs (GOx and HRP) can be rolled into tubular shapes [44]; (c) A swinging arm for cofactor transfer between the enzymes (malate dehydrogenase (MDH) and glucose-6-phosphate dehydrogenase (G6pDH)) assembled on a DNA tile [45]; (d) A modular and tubular DNA origamibased enzyme cascade (GOx and HRP) nanoreactor [46]; (e) An artifical enzyme cascade (xylose reductase (XR) and xylitol dehydrogenase (XDR)) performing a cofactor coupled cascade reaction on DNA origami [47]; (f) An artificial three-enzyme (lactate dehydrogenase (LDH), MDH and oxaloacetate decarboxylase (OAD)) pathway organized using a DNA nanostructure [48]. (a) is reproduced with permission from [43]. Copyright American Chemical Society, 2012. (b) is reproduced with permission from [44]. Copyright American Chemical Society, 2013. (c) is reproduced with permission from [45]. Copyright Nature Publishing Group, 2014. (d) is reproduced with permission from [46]. Published by The Royal Society of Chemistry, 2015. (e) is reproduced with permission from [47]. Copyright American Chemical Society, 2016. (f) is reproduced with permission from [48]. Copyright John Wiley and Sons, 2016. Inspired by the above-mentioned work, Fu et al. [44] designed rectangular (100 nm × 70 nm) DNA tiles with GOx-HRP cascade pairs precisely positioned 15 nm apart from each other. By using sticky-end extensions on the top and bottom edges of the DNA origami rectangles, they induced the tile to form short DNA nanotubes (Figure 3b). Efficiency of the enzyme cascade reaction was quantitatively measured using an excess amount of reactant glucose and the chromogenic reaction of the reporter ABTS2− (substrate for HRP). The activity was highest when the enzymes were located in a confined nanospace within the DNA nanotube. When the enzymes were attached to the semiconfined planar DNA tile, the activity was lower, but still higher than that of free cascade controls, which showed the lowest activity. Hence, these nanoscale bioreactors provide access to an artificial system for studying biological processes in organized cell-mimicking environments. Swinging arms are key constituents of sequenced catalytic transformations in many naturally occurring multi-enzyme complexes. The arm is commonly a chemical group covalently attached to the Figure 3. ( a ) A glucose oxidase (GOx) – horseradish peroxidase (HRP) enzyme cascade pair assembled on a rectangular DNA origami [ 43 ]; ( b ) A rectangular DNA origami shape with attached enzyme cascade pairs (GOx and HRP) can be rolled into tubular shapes [ 44 ]; ( c ) A swinging arm for cofactor transfer between the enzymes (malate dehydrogenase (MDH) and glucose-6-phosphate dehydrogenase (G6pDH)) assembled on a DNA tile [ 45 ]; ( d ) A modular and tubular DNA origami-based enzyme cascade (GOx and HRP) nanoreactor [ 46 ]; ( e ) An artifical enzyme cascade (xylose reductase (XR) and xylitol dehydrogenase (XDR)) performing a cofactor coupled cascade reaction on DNA origami [ 47 ]; ( f ) An artificial three-enzyme (lactate dehydrogenase (LDH), MDH and oxaloacetate decarboxylase (OAD)) pathway organized using a DNA nanostructure [ 48 ]. ( a ) is reproduced with permission from [ 43 ]. Copyright American Chemical Society, 2012. ( b ) is reproduced with permission from [ 44 ]. Copyright American Chemical Society, 2013. ( c ) is reproduced with permission from [ 45 ]. Copyright Nature Publishing Group, 2014. ( d ) is reproduced with permission from [ 46 ]. Published by The Royal Society of Chemistry, 2015. ( e ) is reproduced with permission from [ 47 ]. Copyright American Chemical Society, 2016. ( f ) is reproduced with permission from [ 48 ]. Copyright John Wiley and Sons, 2016. Inspired by the above-mentioned work, Fu et al. [ 44 ] designed rectangular (100 nm ˆ 70 nm) DNA tiles with GOx-HRP cascade pairs precisely positioned 15 nm apart from each other. By using sticky-end extensions on the top and bottom edges of the DNA origami rectangles, they induced the tile to form short DNA nanotubes (Figure 3b). Efficiency of the enzyme cascade reaction was quantitatively measured using an excess amount of reactant glucose and the chromogenic reaction of the reporter ABTS 2´ (substrate for HRP). The activity was highest when the enzymes were located in a confined nanospace within the DNA nanotube. When the enzymes were attached to the semiconfined planar DNA tile, the activity was lower, but still higher than that of free cascade controls, which showed the lowest activity. Hence, these nanoscale bioreactors provide access to an artificial system for studying biological processes in organized cell-mimicking environments. Swinging arms are key constituents of sequenced catalytic transformations in many naturally occurring multi-enzyme complexes. The arm is commonly a chemical group covalently attached to the enzyme complex via a flexible linker that enables the direct transfer of substrate molecules between multiple active sites within the complex. Fu et al. [ 45 ] constructed a DNA nanostructure for assembling a multi-enzyme system that is equipped with an artificial swinging arm. The arm was designed to efficiently channel hydride transfer between two dehydrogenases. The whole design is illustrated in Figure 3c. The nanostructure complex utilized a two-enzyme cascade composed of glucose-6-phosphate dehydrogenase (G6pDH) and malic dehydrogenase (MDH) positioned on a DNA double-crossover (DX) tile scaffold. In the cascade sequence G6pDH catalyzes the oxidation Nanomaterials 2016,6, 139 6 of 16 of glucose-6-phosphate and the reduction of NAD + (nicotinamide adenine dinucleotide, oxidized) to NADH (nicotinamide adenine dinucleotide, reduced). In the second cycle, MDH catalyzes the reduction of oxaloacetate to malic acid using the NADH produced by G6pDH. The swinging arm, an NAD + -equipped poly-thymine (poly-T) oligonucleotide (20 nucleotides long), was adhered to the DNA tile surface exactly halfway between the anchored enzymes G6pDH and MDH. The swinging arm’s capability to boost dehydrogenase activity in complexes containing one enzyme coupled to a single NAD + arm was measured individually in bulk solution for three distances (7, 14 and 21 nm). The highest activity for both G6pDH and MDH was observed at the 7 nm distance showing ca. 25-fold enhancement of activity compared to an enzyme system in the presence of the same concentration (100 nM) of freely diffusing NAD + . In the same experimental conditions, the activity of the fully assembled G6pDH–NAD + –MDH two-enzyme nanostructure with a swinging arm is ca. 90-fold higher than that obtained using the same two-enzyme complex but with freely diffusing NAD+. Linko et al. [ 46 ] designed and fabricated an enzyme reactor, which consists of two distinct tubular 3D DNA origami building blocks with either GOx or HRP enzymes anchored inside the origami compartment through biotin–NeutrAvidin (NTV) binding (Figure 3d). Both units were fabricated separately, and ‘glued’ together via a programmable DNA base-pairing by hybridizing 32 short (three to six bases) sequences. The short sequences that were sticking out at the end of one unit were paired with free scaffold sites located at the edge of another unit. The other end of the origami unit was passivated by overhanging single-stranded poly-T sequences (8 nucleotides) in order to prevent the formation of multimers. The catalytic activity of a two-unit nanoreactor was monitored in the environment containing excess amounts of D-glucose as a reactant and 3,3 1 ,5,5 1 -tetramethylbenzidine (TMB) as a reporter in order to achieve a reaction that is restricted by the diffusion rate of the intermediate product H 2 O 2 . Compared to the control samples (similarly prepared samples but without NTV binding sites for enzymes), the assembled twin-unit nanoreactor has much higher activity, thus indicating that unspecific binding between enzymes and origami structures is insignificant. Ngo et al. [ 47 ] introduced cofactor-coupled cascade reactions on a DNA origami scaffold. The cascade was based on the D-xylose metabolic pathway, and combined two enzymes: xylose reductase (XR) and xylitol dehydrogenase (XDR). The enzymes were attached to the DNA scaffold with DNA-binding protein adaptors, the zinc finger protein (zif268) and the basic leucine-zipper protein (GCN4). The cascade mechanism relies on the recycling of cofactor NADH between the enzymes, which is possible due to their close proximity. Within the metabolic pathway of xylose, the first enzyme XR converts xylose into xylitol by consuming the cofactor NADH. The produced xylitol and NAD + are both simultaneously transported to the second enzyme XDH, which converts xylitol into xylulose by consuming NAD+to recycle the NADH cofactor (Figure 3e). Liu et al. [ 48 ] assembled an artificial three-enzyme pathway on a series of DNA nanoscaffolds in order to study the dependence of their activities. They measured the activities of an MDH-OAD-LDH (malate dehydrogenase–oxaloacetate decarboxylase–lactate dehydrogenase) cascade with variable spatial distances and geometric arrangements. The three-enzyme pathway (Figure 3f) starts with the MDH-catalyzed oxidation of malic acid to oxaloacetate (OAA) and the simultaneous reduction of NAD + to NADH. In the next cycle OAD converts OAA into pyruvic acid and inhibits its conversion back to malic acid. In the third cycle LDH consumes the reduced NADH and pyruvic acid to produce lactic acid. Unlike the above-mentioned two-enzyme systems, the overall activity of the three-enzyme pathway was more dependent on the geometric patterns that arranged enzymes within a short distance (10–30 nm) of each other rather than with interenzyme spacings. By optimizing the geometric patterns of the three enzymes, a five-fold activity enhancement was obtained compared to the unassembled free enzymes. In addition, the depletion of the pathway intermediates was very efficient in the assembled enzyme systems with little detectable NADH in the bulk solution, indicating that nearly all NADH was coupled into the enzyme pathway without leakage. Nanomaterials 2016,6, 139 7 of 16 4. Enzymatic Nanodevices with Motion Besides the static nanoreactors discussed in the previous section, there are compelling examples of in vitro nanodevices that can control enzyme activity. These devices can be switched between an active and inactive state by introducing a specific trigger. The triggers are usually DNA strands that are able to perform preprogrammed strand displacement reactions. Alternatively, some of the systems can autonomously regulate the reaction(s). Here, a few examples of mechanical regulatory DNA-enzyme devices, autonomous molecular systems and their working principles are reviewed (see also Table 2). Table 2. Examples of mechanical regulatory DNA-enzyme devices. Type Function Key Aspects DNA nanotweezers [49–52] equipped with cascade pairs or with the enzyme and its cofactor. The tweezers can be opened and closed through a strand-displacement reaction. The enzyme activity can be controlled by switching the tweezers reversibly. A tubular DNA origami nanoreactor [53]. The lid of the tube can be opened and closed with the help of lock and key strands. Flowthrough of the compounds into the confined reaction chamber is controlled by the lid. A four-arm DNA origami nanoactuator [54]. A distance change in a driver site can be propagated to the mirror site containing binding sites for cargo molecules. The actuator can be driven using different mechanisms, and it can be used for, e.g., tuning fluorescence behavior of enhanced fluorescent protein (eGFP). Aptamer-based logical circuit [ 55 ]. The autonomous logical circuit controls α-thrombin activity through the convertor, controller and generator modules. α-thrombin aids blood coagulation, and therefore systems such as this may find intriguing biomedical uses. 4.1. Mechanical Regulatory DNA-Enzyme Devices Liu et al. [ 49 ] employed a DNA tweezer nanostructure to actuate the reaction between a G6pDH/NAD + enzyme-cofactor pair. In this construct (Figure 4a), the enzyme and cofactor were attached to two different ca. 14-nm-long arms. Actuation of the enzyme function was achieved by switching between open and closed states of the tweezers, in other words by spatially separating the enzyme-cofactor pair for inhibition or bringing the pair together for activation, respectively. In the reaction cycle, NAD + is first reduced to NADH by G6pDH. Then, phenazine methosulfate (PMS) catalyzes electron transfer from NADH to resazurin, which produces strongly fluorescent resorufin. In the tweezer geometry, a 25-nucleotide (nt) ssDNA oligomer connected the ends of the tweezer arms and served as a structural regulatory element to control the state of the system. The open state can be attained by disrupting the hairpin via hybridization between a complementary set strand and a hairpin, thus generating a rigid ca. 16-nm-long dsDNA domain between the ends of the tweezer arms. By adding a fuel strand (fully complementary to the set strand) to the system, a hairpin is released by a strand-displacement mechanism and the tweezers are switched back to the closed state. Opening and closing mechanisms have been further optimized by Dhakal et al. [50]. Moreover, Xin et al. [ 51 ] used similar nanotweezers and chose the GOx-HRP cascade as a model to demonstrate the reversible regulation of the enzyme cascade reaction. The DNA machine was comprised of double-crossover (DX) motifs, which formed two rigid arms (glued together by an immobile four-way junction). A DNA motor, which can switch between a stem-loop and a double-helix structure driven by a strand displacement reaction, was incorporated into the middle of the DNA machine to cycle between open and closed states. This kind of device could also be used to reversibly regulate the target binding affinity of a thrombin protein, as shown by Chou et al. [52]. Nanomaterials 2016,6, 139 8 of 16 Nanomaterials 2016, 6, 139 8 of 16 DNA machine to cycle between open and closed states. This kind of device could also be used to reversibly regulate the target binding affinity of a thrombin protein, as shown by Chou et al. [52]. Figure 4. (a) Nanotweezers to regulate enzyme activity [49]; (b) Tubular nanoreactor with switchable lid to control the flowthrough of the reaction compounds [53]; (c) DNA origami nanoactuator that can be driven by, e.g., single-stranded DNA (ssDNA) strands or restriction enzymes [54]; (d) Aptamerbased logical molecular circuit to control thrombin activity [55]. (a) is reproduced with permission from [49] Copyright Nature Publishing Group, 2013. (b) is reproduced with permission from [53]. Copyright The Royal Society of Chemistry, 2016. (c) is reproduced with permission from [54]. Published by Nature Publishing Group, 2016. (d) is reproduced with permission from [55]. Copyright American Chemical Society, 2012. Wang et al. [53] prepared a DNA origami nanochannel as a scaffold for monitoring the GOxHRP cascade reaction. The channel, 100 nm in length and 22 nm in diameter, was formed by rolling up a rectangular origami object with the help of sticky ends, placed as extensions at the top and bottom helices of the sheet-like structure (depicted in Figure 4b). A row of 11 staple strands, called shutter strands, which contain 15 nucleotides long overhangs in an upright position to the concave side, formed a shutter at the end of the nanochannel which can control the opening and closing of the channel upon stimuli. By adding the ‘lock strands’, i.e., ssDNA molecules complementary to the 15 Figure 4. ( a ) Nanotweezers to regulate enzyme activity [ 49 ]; ( b ) Tubular nanoreactor with switchable lid to control the flowthrough of the reaction compounds [ 53 ]; ( c ) DNA origami nanoactuator that can be driven by, e.g., single-stranded DNA (ssDNA) strands or restriction enzymes [ 54 ]; ( d ) Aptamer-based logical molecular circuit to control thrombin activity [ 55 ]. ( a ) is reproduced with permission from [ 49 ] Copyright Nature Publishing Group, 2013. ( b ) is reproduced with permission from [ 53 ]. Copyright The Royal Society of Chemistry, 2016. ( c ) is reproduced with permission from [ 54 ]. Published by Nature Publishing Group, 2016. ( d ) is reproduced with permission from [ 55 ]. Copyright American Chemical Society, 2012. Wang et al. [ 53 ] prepared a DNA origami nanochannel as a scaffold for monitoring the GOx-HRP cascade reaction. The channel, 100 nm in length and 22 nm in diameter, was formed by rolling up a rectangular origami object with the help of sticky ends, placed as extensions at the top and bottom helices of the sheet-like structure (depicted in Figure 4b). A row of 11 staple strands, called shutter strands, which contain 15 nucleotides long overhangs in an upright position to the concave side, formed a shutter at the end of the nanochannel which can control the opening and closing of the channel upon stimuli. By adding the ‘lock strands’, i.e., ssDNA molecules complementary to the 15 nt overhangs, rigid DNA duplexes were formed, resulting in an efficient closing of the shutter. Reopening the nanochannel was achieved by using fully complementary ‘key strands’. The key strands hybridized with the lock strands, displacing them from the channel and therefore opening the shutter. 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