Aptamer-Functionalized Hybrid Nanostructures for Sensing, Drug Delivery, Catalysis and Mechanical Applications

Sequence-specific nucleic acids exhibiting selective recognition properties towards low-molecular-weight substrates and macromolecules (aptamers) find growing interest as functional biopolymers for analysis, medical applications such as imaging, drug delivery and even therapeutic agents, nanotechnology, material science and more. The present perspective article introduces a glossary of examples for diverse applications of aptamers mainly originated from our laboratory. These include the introduction of aptamer-functionalized nanomaterials such as graphene oxide, Ag nanoclusters and semiconductor quantum dots as functional hybrid nanomaterials for optical sensing of target analytes. The use of aptamer-functionalized DNA tetrahedra nanostructures for multiplex analysis and aptamer-loaded metal-organic framework nanoparticles acting as sense-and-treat are introduced. Aptamer-functionalized nano and microcarriers are presented as stimuli-responsive hybrid drug carriers for controlled and targeted drug release, including aptamer-functionalized SiO2 nanoparticles, carbon dots, metal-organic frameworks and microcapsules. A further application of aptamers involves the conjugation of aptamers to catalytic units as a means to mimic enzyme functions “nucleoapzymes”. In addition, the formation and dissociation of aptamer-ligand complexes are applied to develop mechanical molecular devices and to switch nanostructures such as origami scaffolds. Finally, the article discusses future challenges in applying aptamers in material science, nanotechnology and catalysis.

The last two decades have introduced many different nanomaterials exhibiting unique optical, catalytic, physical adsorption and porosity properties derived from the nanodimensions and high surface area of the nanostructures. For example, the plasmonic and interplasmonic coupling characteristic to noble metal nanoparticles, such as Au or Ag nanoparticles, the catalytic and electronic properties of metal nanoparticles or carbon nanotubes and the size-controlled luminescence properties of semiconductor quantum dots, e.g., CdSe or CdTe, have been extensively used to develop many different sensing devices and imaging methods [80][81][82][83]. Not surprisingly, the integration of aptamers with nanomaterials led to the generation of aptamer-nanomaterial hybrid systems that combine the specific recognition functions of aptamers and the unique physical properties of nanomaterials. For example, the integration of aptamers with plasmonic Au nanoparticles allowed the sensing of Hg(II) [84][85][86] or thrombin [25].
In the present perspective article, we present a summary of examples originating from our laboratory demonstrating the broad applicability of aptamer-functionalized hybrid nanostructures in various scientific disciplines. Our aim is to highlight the role of aptamernanomaterial hybrids in the general topic of nanobiotechnology. Specific applications of aptamer-nanomaterial hybrids in developing sensors, drug-delivery carriers, new catalytic materials and supramolecular machines will be addressed.

Hybrid Aptamer Nanostructures for Sensing Applications
The sequence-specific binding properties of aptamers have been widely applied to develop sensing platforms [12][13][14][15]. By the integration of aptamers with nanomaterials, hybrid optical sensing platforms that combine the specific recognition properties of aptamers with the unique optical or physical properties of nanomaterials were assembled [87][88][89]. This topic is introduced in this section with the integration of silver nanoclusters (AgNCs), semiconductor nanoparticles and graphene oxide.
Single stranded nucleic acids adsorb strongly to graphene oxide nanosheets. Accordingly, aptamers were functionalized with fluorophores and adsorbed onto graphene oxide nanosheets. The fluorophore labels were quenched by the graphene oxide, yet desorption of the aptamers from the graphene oxide carrier, through the formation of aptamer-ligand complexes, switched on the fluorescence of the labels, thus allowing the optical detection of the ligands. This is exemplified in Figure 1A with the multiplexed analysis of thrombin and ATP by the respective aptamers [90]. The ATP aptamer was labeled with the carboxy-X-rhodamine (ROX) fluorophore (λ em = 603 nm), whereas the thrombin aptamer was labeled with the 6-carboxyfluorescein (FAM) fluorophore (λ em = 518 nm). The labeled aptamers were adsorbed onto graphene oxide and their fluorescence was quenched. The multiplexed analysis of the ATP and thrombin analytes is presented in Figure 1B. While in the absence of the analyte no fluorescence in the solution was detected (Panel I). In the presence of thrombin the fluorescence of FAM was switched on in the solution (Panel II), and in the presence of ATP the fluorescence of ROX was switched on (Panel III). In the presence of ATP and thrombin, both aptamer-ligand complexes were desorbed into the solution resulting in the fluorescence of FAM and ROX.  [90]. Copyright 2012 American Chemical Society. Arrows: the arrows correspond to typical excitation and emission arrows. In a related system, the fluorescent properties of sequence-specific nucleic acidstabilized silver nanoclusters (AgNCs) provided means to design optical aptosensors [91]. Nucleic acid-stabilized AgNCs, λ em = 616 nm, were extended by the ATP aptamer sequence to yield a functional probe for sensing ATP. The probe was adsorbed onto graphene oxide resulting in the quenching of the fluorescence of the AgNCs. In the presence of ATP, the probe was desorbed from the graphene oxide support leading to the switching on of fluorescence of the NCs (λ = 616 nm) (Figure 2A). The fluorescent spectra of the desorbed AgNCs-ATP aptamer/ATP complex at different concentrations of ATP, and the resulting calibration curve, are displayed in Figure 2B. The sensing of ATP by the hybrid nucleic acid/AgNCs/graphene oxide was specific and the sensing matrix did not respond to other nucleotide triphosphates. The sensing platform was, similarly, applied to sense thrombin using a different-sized nucleic acid-stabilized fluorescent AgNCs (λ = 775 nm) conjugated to the thrombin aptamer and adsorbed onto graphene oxide. (A) Application of graphene oxide modified with ATP aptamer conjugated to nucleic acidstabilized silver nanoclusters (AgNCs) as a hybrid system for the analysis of ATP. The fluorescence of the AgNCs is quenched in the aptamer/graphene oxide hybrid structure and it is switched on upon the desorption of the AgNCs-ATP aptamer/ATP complex from the graphene oxide support. (B) Fluorescence spectra generated upon desorption of the AgNCs-ATP aptamer/ATP complex in the presence of different concentrations of ATP for a fixed time-interval of 1 h. a = 0 mM; b = 0.025 mM; c = 0.05 mM; d = 0.25 mM; e = 0.5 mM; f = 1 mM; g = 2 mM; h = 3 mM. Inset: derived calibration curve. Reproduced with permission from ref. [91]. Copyright 2013 American Chemical Society. Arrows: the arrows correspond to typical excitation and emission arrows.
Another optical nanostructure-based aptasensor is displayed in Figure 3 [92]. Figure 3A depicts the chemiluminescence resonance energy transfer (CRET)-stimulated sensing of ATP using a supramolecular hemin/G-quadruplex-ATP aptamer-CdSe semiconductor quantum dot (QD) hybrid module. The sensing platform made use of the chemiluminescence generated by the hemin/G-quadruplex catalyzed oxidation of luminol by H 2 O 2 and the subsequent CRET process to the CdSe QDs that leads to the luminescence of the QDs without external irradiation. Accordingly, CdSe QDs were modified with the nucleic acid sequence (1) that included the ATP aptamer subunit, X a , and the G-quadruplex subunit, Y a . In the presence of the coadded strand (2), composed by the complementary ATP aptamer sequence, X b , and the G-quadruplex subunit, Y b , and ATP analyte, K + -ions and hemin, the cooperative stabilization of the supramolecular ATP/ATP aptamer complex-hemin/Gquadruplex on the QDs carrier occurred. The hemin/G-quadruplex-catalyzed oxidation of luminol by H 2 O 2 generated chemiluminescence stimulating the CRET process to the QDs and the consequent luminescence of the QDs at λ = 620 nm. As the efficiency of the CRET process was controlled by the concentration of ATP forming the supramolecular catalytic nanostructure on the QDs, the resulting CRET signal allowed the quantitative analysis of ATP, Figure 3B,C. A different supramolecular nanostructure for the optical detection of aptamer-ligand complexes included DNA tetrahedra nanostructures. DNA three dimensional (3D) tetrahedra nanostructures have attracted substantial recent interest in DNA nanotechnology. The ease to self-assemble different-sized DNA tetrahedra structures with high yields from preengineered single strands exhibiting appropriate complementarities provides versatile 3D structures for different applications [93][94][95]. The integration of DNAzymes into the edges of the tetrahedra, the tethering of nucleic acids to the corners of the tetrahedra nanostructures [96] and the demonstration of the superior cell permeation [97][98][99] provide versatile methods for cell targeting and recognition events. Indeed, the DNA tetrahedra nanostructures were extensively used for biomedical applications, including the development of sensors and cell imaging [100,101]. Here, we present the application of an aptamerfunctionalized supramolecular DNA tetrahedron nanostructure as a functional module for the multiplexed analysis of aptamer-ligand complexes ( Figure 4A) [102]. A DNAtetrahedron module, I, consisting of edges x, y, z stabilized by three spatially-separated fluorophore-quencher pairs (fluorophores = FAM/quencher BHQ1 edge x; ROX/quencher BHQ2 edge y and Cy5/quencher BHQ2 edge z) was prepared. The edges were stretched by hybridization with the respective aptamer, ATP aptamer (x') VEGF (vascular endothelial growth factor) aptamer (y') and thrombin aptamer (z'). In the spatially-aptamer stretched tetrahedron module the fluorescence of the respective fluorophores was switched on. Subjecting the sensing module to any of the aptamer ligands (ATP, VEGF, thrombin) resulted in the displacement of the respective aptamer-ligand complexes and in the reconfiguration of the edges into hairpin structures, H a , H b or H c , where the fluorophore-quencher pairs are forced into an intimate position leading to the quenching of the respective fluorophore. Figure 4B exemplifies the sensing of variable concentrations of VEGF by the sensing module. Similar selective sensing of ATP and thrombin were demonstrated by the reconfiguration of the edges x or z in the presence of ATP or thrombin, respectively. In addition, the DNA tetrahedron sensing module was successfully applied for the multiplex analysis of the different analytes, Figure 4C. The use of aptamers as therapeutic materials was further addressed by the incorporation of aptamers in stimuli-responsive nanocarriers that allow the triggered release of the aptamer by appropriate cellular conditions. This is exemplified in Figure 5 with the assembly of ZIF-8 metal-organic framework nanoparticles (NMOFs), loaded with glucose oxidase (GOx) and the VEGF aptamer [103]. The ZIF-8 NMOFs, composed of Zn 2+ -ions interbridged by 2-methylimidazole, are pH-sensitive, and at pH < 5.5 are degraded. Thus, subjecting the GOx/VEGF-loaded NMOFs to glucose results in the aerobic GOx-catalyzed oxidation of glucose to gluconic acid that acidifies the metal-organic framework. The acidic pH leads to the degradation of the NMOFs and the release of the VEGF aptamer that acts as an inhibitor of angiogenesis promoted by VEGF. The integration of the GOx/VEGF aptamer in the NMOFs was confirmed by labeling of the VEGF aptamer with Cy3 and GOx with coumarin and imaging the NMOFs by confocal fluorescence microscopy ( Figure 5B). The glucose-stimulated release of the VEGF aptamer was controlled by the concentration of glucose and, as the concentration of glucose increased, the release of the VEGF aptamer was enhanced ( Figure 5C). This suggests the potential application of the GOx/VEGF-loaded NMOFs for ocular therapeutic treatment via the dose-controlled inhibition of the VEGFinduced angiogenesis blood vessel clotting in the eyes. That is, at high intraocular glucose concentration, the degradation of the NMOFs is enhanced leading to the effective release of the VEGF aptamer and inhibition of VEGF. Nonetheless, at a lower concentration of glucose, the local pH changes in the NMOFs are readily dissipated by the buffered solution of the eye containment leading to the blockage of ZIF-8 decomposition and to the release of the aptamer. Thus, the intraocular concentration changes could be envisaged as triggers for the dose-controlled release of the VEGF aptamer in the eye, Figure 5D.
Nucleic acid structures have been often used as caging units of drug carriers that can be unlocked by nucleic acid biomarkers such as miRNAs [137], the pH-induced separation of DNA locks via the dissociation of i-motifs [138,139] or triplexes, [140,141] the separation of K + -ion stabilized G-quadruplex locks by crown ether [142,143], and the degradation of DNA locks by enzymes [144] or DNAzymes [145]. Physical triggers, such as heat [146][147][148] or light [149][150][151][152][153] have also been used to uncage DNA-gated carriers. The sequence-specific recognition properties of aptamer-ligand complex have been extensively used to design nucleic acid-based gating units being unlocked through the formation of aptamer-ligand complexes [154]. These unlocking principles have specific merit since many ligands act as biomarkers for diseases, and then the biomarker-induced uncaging of the carriers may act as autonomous sense-and-treat systems for the target-controlled release of drugs. Here, we provide several examples of aptamer-gated carriers for the controlled release of drugs.
Metal-organic framework nanoparticles (NMOFs) provide a broad class of porous materials with high loading capacity of drugs [107]. Figure 6 exemplifies the stepwise synthesis of the NMOFs and their gating with ATP aptamer locks [155]. Azide-modified UiO-68 NMOFs were functionalized with DBCO-modified nucleic acid (3) (DBCO = dibenzocyclooctyne) using the click chemistry principle. The NMOFs were loaded with the anticancer drug doxorubicin (DOX) and the loaded NMOFs were locked by hybridization with the ATP aptamer (4).   [155]. In one configuration, path I, subjecting the NMOFs to the ATP ligand results in the formation of the ATP/ATP aptamer complexes and the release of the drug. In the second configuration, path II, the ATP aptamer (x) is further elongated with the AS1411 aptamer sequence (y), that binds to the nucleolin receptor associated with different cancer cells as a targeting element, sequence (5). Figure 7B   Other aptamer-gated NMOFs were developed following the protocol previously described including VEGF aptamer-functionalized DOX-loaded NMOFs for the release of a drug by over-expressed VEGF in cancer cells, [156] and thrombin aptamer-functionalized Apixaban-loaded NMOFs for the thrombin triggered release of the antiblood-clotting drug through the formation of the thrombin/thrombin aptamer complexes [157].
In a further approach [145], the UiO-68 NMOFs loaded with DOX were locked with duplex nucleic acids (6)/(7), where the strand (7) includes a loop domain composed of the ATP aptamer sequence (light blue) separated by two subunits (dark blue) that correspond to the Mg 2+ -ion-dependent DNAzyme sequence, and the strand (6) includes the ribonucleobase containing sequence that corresponds to the substrate of the Mg 2+ -ion-dependent DNAzyme. The flexibility of the loop region of (7) prohibits the formation of the active Mg 2+ -ion-dependent DNAzyme. In the presence of ATP, the loop domain reconfigures into the ATP/ATP aptamer ligand complex that results in the rigidification of loop region and the active Mg 2+ -dependent DNAzyme structure. This leads to the ATP-triggered activation of the DNAzyme that catalyzes the cleavage of the substrate, the sequestered dissociation of the locks and the release of the loaded drug ( Figure 8A). Figure 8B reveals that the carrier is unlocked only in the presence of ATP and Mg 2+ -ions that act as cooperative triggers for the release of the drug. Cytotoxicity experiments revealed that the over-expressed ATP in MDA-MB-231 breast cancer cells stimulated ca. 45% cell death after treatment of the cells with the DOX-loaded (6)/(7)-locked NMOFs while normal epithelial breast cells were unaffected under similar conditions ( Figure 8C). Similar cytotoxic effects were observed upon following colorimetrically the time-dependent apoptosis of spheroid MDA-MB-231 aggregates and appropriate control systems ( Figure 8D). The cytotoxicity and downstream in vivo degradation of NMOFs and consequent release of metal ions and bridging ligands were addressed in several studies [158,159]. Although these effects should be examined for each specific NMOF carrier, the available reports claimed minute toxicity of the particles or degraded metal ions/ligands (e.g., ZIF-8 NMOFs). Mesoporous SiO 2 nanoparticles (MP SiO 2 NPs) provide a versatile carrier for drug delivery [160]. This is exemplified in Figure 9A with the loading of the nucleic acid (8)functionalized MP SiO 2 NPs with the anticancer drug camptothecin, CPT [161]. The heated (8)-functionalized NPs (to retain the strand in an open flexible configuration) were loaded with CPT and the cooled-down of the carriers resulted in the reconfiguration of (8) into a hairpin structure that locked the drug in the pores of the NPs. The hairpin loop structure and part of the stem domain were engineered to include the ATP aptamer sequence. In the presence of ATP, the hairpin structure was reconfigured to yield the ATP/ATP aptamer complex in a hairpin structure that includes the 3 -end of the aptamer in a fully hybridized structure with the counter stem domain. In the presence of Exonuclease III, Exo III, the hairpin stem domain is hydrolytically digested, resulting in the uncaging of the hairpin locks, leading to the release of ATP and CPT. The Exo III cleavage of the hairpin structure provides a path to regenerate ATP for further unlocking events. Figure 9B  Carbon nanomaterials, such as carbon nanotubes, graphene oxide or porous carbon nanoparticles have attracted substantial interest as functional materials for different applications [162][163][164]. Within this class of nanomaterials, carbon nanoparticles, C-dots, are of particular interest due to their high surface area, surface functionalities, allowing the binding of ions or ligands, and photophysical luminescence functions [165]. These properties allowed the use of C-dots as catalysts [166] or sensors [167,168]. In addition, the modification of C-dots, their low cytotoxicity and effective cell permeation properties allowed their use as drug carriers [169]. As aptamers are promising modules for therapeutic applications, the use of C-dots as a support for carrying aptamers seems an attractive path to apply aptamer/C-dots hybrids for drug delivery and therapeutic applications. This is exemplified with the application of C-dots modified with the VEGF aptamer for topical treatment of ocular disorders [170]. VEGF induces angiogenetic driven diseases associated with age-related macular degeneration (AMD) and diabetic retinopathy (DR), causing blindness. The inhibition of the VEGF angiogenetic process through binding of VEGF to the VEGF aptamer is a recently viable therapy yet limited by the need of repeated intraocular injections. Figure 10A depicts the modification of carboxylic acidfunctionalized C-dots with the VEGF aptamer. The C-dots were covalently modified with the amine-functionalized nucleic acid (9) to which the VEGF aptamer (10) was hybridized. This hybrid carrier was designed as a functional platform for carrying the aptamer across the corona, into the retina and posterior sclera, where the VEGF-induced release of the aptamer is anticipated to form the G-quadruplex VEGF aptamer-VEGF complex, leading to the inhibition of the protein. Optical measurements that followed the fluorescence of the C-dots demonstrated the effective permeation of the (9)/(10)-functionalized C-dots across the various eye structures and their accumulation in the retina and choroid ( Figure 10B). In addition, no cytotoxic effects of the C-dots were observed and the inhibition function of the aptamer/C-dots hybrid was demonstrated using an in vitro model of choroidal vascular angiogenesis ( Figure 10C). The VEGF aptamer-loaded C-dots revealed an obvious inhibitory effect on choroidal blood vessel growth and sprouting as compared to nontreated samples, and their effect was comparable to commercial therapeutic drugs administered, at present, by invasive injection. That is, the aptamer/C-dots hybrid has the potential of topical administration to resolve a major problem in diseases. A further versatile aptamer-responsive, therapeutically-promising drug carrier system includes aptamer-gated hydrogel microcapsules [171]. Figure 11A depicts the construction of drug-loaded microcapsules and the schematic unlocking of the microcapsules through the formation of aptamer-ligand complexes, where the ligand is a biomarker for a disease. Calcium carbonate microparticles were impregnated with DOX-dextran (DOX-D) as an anticancer drug. The DOX-D-loaded CaCO 3 microparticles were coated with positively charged polyallylamine hydrochloride (PAH). Polyacrylic acid (PAA) modified with the amino nucleic acid (11) was coadsorbed by electrostatic interactions onto the positively charged PAH-coated particles. The polyacrylamide polymer chains P A and P B were functionalized with hairpin H A , (12), and H B (13) linked to the polymer through a short tether (14) (for directionality reasons). The hairpin H A includes in its stem region and the loop domain the ATP aptamer sequence and, in addition, a part of the loop domain includes the complementary sequence for the promoter (11) (12) and (13). Subsequently, the CaCO 3 core microparticles were dissolved in the presence of EDTA to yield DOX-D-loaded microcapsules. As the duplex (12)/(13) bridging units include the ATP aptamer sequence, in the presence of ATP, the duplex units are separated by forming the (12)-ATP aptamer/ATP (ligand) complexes, resulting in the perforation of the hydrogel coating and the release of the DOX-D-loaded drug ( Figure 11B). The resulting DOX-D-loaded microcapsules before and after etching of the CaCO 3 cores were imaged by scanning electron microscopy (SEM) ( Figure 11C) and confocal fluorescence and brightfield microscopy ( Figure 11D). Figure 11E shows the ATP-stimulated unlocking of the microcapsules, while in the absence of ATP the release of DOX-D is effectively blocked, uncaging of the microcapsules and release of DOX-D is observed in the presence of ATP. The release of ATP is controlled by the concentration of ATP, and as the concentration of ATP increased the release process was enhanced. Furthermore, control experiments reveal specificity toward ATP as uncaging agent, and other nucleotide triphosphate (CTP, GTP, TTP) did not affect the release process. As ATP is over-expressed in cancer cells, it could act as trigger to release the drug in cancer cells. Indeed, cytotoxicity experiments evaluating the effect of the DOX-D-loaded microcapsules on MDA-MB-231 breast cancer cells and control MCF-10A normal epithelial breast cells revealed obvious cytotoxic efficacy of the microcapsules towards the cancer cells ( Figure 11F). Treatment of the MDA-MB-231 cancer cells and the MCF-10A normal cells with the DOX-D-loaded microcapsules resulted in 35% cell death of the cancer cells and only 10% cell death of the normal cells after five days of treatment. The superior cytotoxicity of the microcapsules towards the cancer cells was attributed to the elevated contents of ATP in the cancer cells and to the improved permeation of the microcapsules into the cancer cells as compared to the normal cells. Although the origin for the superior permeation of the microcapsules into the cancer cells is, at present, not fully understood, we believe that the enhanced porosity of the cancer cell membrane facilitates the enhanced permeability and retention effect (EPR) transport through the cancer cell boundaries.

Aptamer Nanostructures for Catalysis
The selective binding properties of aptamers have been utilized to design hybrid nanostructures mimicking native enzymes-nucleoapzymes [7]. The covalent conjugation of a catalytic unit to sequence-specific aptamers provides means to duplicate the cooperative functions of the catalytic sites and binding sites in native enzymes, where the substrate acts as the ligand that binds to the aptamer. As the catalyst may be conjugated to the 3 -end, 5 -end or middle portions of the aptamer, or the catalytic unit may be linked to the 3 -or 5 -ends by different space elements, one may envisage the possibility to synthesize sets of catalytic-aptamer nucleoapzyme module exhibiting variable activities ( Figure 12). Thus, it would be interesting to computationally elucidate the structure-catalytic function relationships with the set of nucleoapzymes and compare the computation features to the experimental results with the vision that in silico design of superior nucleoapzymes could be accomplished in the future. The molecular engineering of the nucleoapzymes involves several stages. (i) One must define the catalytic transformation for which the nucleoapzymes should be designed. (ii) The availability of an aptamer for the reaction substrate is essential. This is elicited by the well-established SELEX procedure. (iii) A catalyst for the target chemical transformation should be selected. As many catalytic nucleic acid modules, DNAzymes, are available, they may be conjugated as a part of the nucleic acid sequence to yield the nucleoapzymes. Alternatively, a catalytic ligand, e.g., imidazole, or a transition metal complex catalyst, capable of catalyzing the target chemical transformation, should be covalently tethered to the aptamer sequence to yield the respective nucleoapzymes. The catalytic performance of the respective nucleoapzymes should then be compared to control systems that include the separated aptamer/catalytic site components and to a system comprising the catalytic site covalently linked to a nucleic acid composed of a scrambled, randomized sequence of the aptamer bases. In addition, the kinetic features of the nucleoapzymes should be analyzed by enzyme kinetic models, and the binding properties of the substrates to the respective nucleoapzyme structures should be evaluated, aiming to identify the structure/function relationships in the respective catalytic modules.
In the past few years, our laboratory has developed the concept of nucleoapzymes, and two examples are introduced. The first example includes the conjugation of the hemin/Gquadruplex DNAzyme catalytic units to the dopamine aptamer to yield a series of nucleoapzymes catalyzing the H 2 O 2 -mediated oxidation of dopamine to dopachrome [172]. Four nucleoapzyme structures are exemplified in Figure 13A. The nucleoapzyme in configuration I includes the catalytic hemin G-quadruplex linked to the 5 -end of the aptamer through a single adenine (A) spacer. The nucleoapzyme in configuration II includes the hemin G-quadruplex conjugated to the 5 -end of the aptamer through a TATA spacer, and the nucleoapzyme in configuration III includes the hemin G-quadruplex linked to the 3 -end of the aptamer using the TATA spacer unit. In nucleoapzyme in configuration IV the hemin G-quadruplex is conjugated to the 5 -end and 3 -end of two aptamer sequences using single adenosine spacer units (A). Figure 13B depicts the kinetic features of the four configurations as compared to the separated hemin/G-quadruplex units, by providing the rates of dopachrome formation in the presence of variable concentrations of dopamine and an excess of the oxidizing agent H 2 O 2 . An additional control experiment includes a structure where the hemin/G-quadruplex catalyst is conjugated to the 5 -end of a nucleic acid composed of a scrambled, randomized sequence of the bases comprising the dopamine aptamer sequence ( Figure 13B, curve (c)). The results demonstrated that all nucleoapzyme, structures I-IV, revealed substantial higher activities as compared to the separated hemin G-quadruplex and aptamer units. For the single hemin G-quadruplex-aptamer conjugates, the catalytic activity of the nucleoapzymes follows the order II > I > III. The nucleoapzyme in configuration II reveals a 20-fold enhanced catalytic activity compared to the separated units. All nucleoapzymes revealed a Michalis-Menten kinetic behavior and reached saturation kinetics consistent with the saturation of the aptamer binding sites by the dopamine substrate. Several conclusions were derived from these kinetic analyses and additional supporting experiments. (i) The nucleoapzyme in configuration II demonstrated superior catalytic properties as compared to configuration I and III (V max = (13.5 ± 0.5) nm s −1 ; k cat /k M = 14.1 × 10 −3 s −1 mM. (ii) The hemin G-quadruplex linked to the scrambled sequence revealed a slight catalytic activity as compared to the separated units, attributed to the electrostatic attraction of the positively charged dopamine substrate to the negatively charged nucleic acid scaffold. (iii) The nucleoapzyme in configuration IV revealed superior catalytic properties due to the concentration of the reaction substrate by two aptamer sequences in the proximity of the catalytic site. (iv) The binding affinity of dopamine to the different nucleoapzymes, evaluated by fluorescence anisotropy measurements, revealed very similar dissociation constants implying that the differences in the catalytic performance of the nucleoapzymes did not originate from binding affinities to the aptamer receptors, suggesting that the different special orientation of the substrate in respect to the active catalytic sites provided the origin for the observed catalytic performance of the structures. To account for the structural features dictating the catalytic performance of the nucleoapzymes, we applied molecular dynamic (MD) simulations to probe the structural features of the nucleoapzymes [173][174][175][176] (Figure 13C). The MD simulations revealed that the hemin/G-quadruplex catalytic site is closer (3-9 nm) to the dopamine binding site in configuration II, as compared to the longer spatial separation of the catalytic site from the substrate binding site in configuration III (9-15 nm).
The second example for constructing nucleoapzymes includes the modification of the aptamer with a transition metal complex as catalytic unit. This is introduced by describing a set of nucleoapzymes mimicking the catalytic activity of ATPase; the hydrolysis of ATP to ADP [177]. A set of nucleoapzymes consisting of the ATP aptamer modified with the bis-Zn 2+ pyridyl-salene complex was synthesized, (Figure 14). This included the linkage of the Zn 2+ -pyridyl-salene complex directly to the 3 -and 5 -ends of the ATP-aptamer, (configuration I and II), and the nucleoapzymes composed of the bis-Zn 2+ -pyridyl-salene complex linked to the 3 -and 5 -ends of the ATP aptamer using a two thymine spacer (2× T), (configurations III and IV, respectively). Figure 14B depicts the kinetic features corresponding to the hydrolysis of ATP to ADP by the nucleoapzyme III, curve (a) and nucleoapzyme IV, curve (b). While no hydrolysis of ATP could be detected in the presence of the separated bis-Zn 2+ -pyridyl-salene complex and ATP aptamer, the catalytic properties of all nucleoapzymes were demonstrated, following the order III > I > IV > II. For example, for nucleoapzyme in configuration III, k cat = 688 × 10 −2 min −1 and K M = 38 ± 7 µM, whereas for nucleoapzyme in configuration IV, k cat = 297 × 10 −2 min −1 K M = 33 ± 6 µM. As the binding affinities of ATP to the different nucleoapzymes were similar, K d = 19 µM, it was suggested that the spatial positioning of ATP in the aptamer receptor scaffold, with respect to the catalyst, is favored in nucleoapzyme III compared to nucleoapzyme IV. Indeed, MD simulations indicated that the catalytic site in the nucleoapzyme in configuration III is positioned in a sterically favored configuration with respect to the hydrolytic reaction site, compared to the spatial separation of the catalytic site from the reaction site in the nucleoapzyme in configuration I (Figure 14). While the distance separating the catalytic site from the reaction site in configuration III is 18Å, the distance in configuration IV is 44Å. The concept of nucleoapzymes was further extended to include Cu(II)-or Fe(III)terpyridine-functionalized dopamine aptamer for the catalyzed H 2 O 2 -mediated oxidation of dopamine [178] and the Fe(III)-terpyridine-modified tyrosinamide aptamer for the oxidative oxygen insertion into tyrosinamide [65]. The functionalization of the cholic acid aptamer with the imidazole ligand led to the catalyzed hydrolysis of a cholic acid ester substrate [179].
The triggered formation and dissociation of aptamer-ligand complexes provides a general means to construct DNA machine-like devices. This is exemplified in Figure 15A with the assembly of a DNA tweezer triggered by ligand-aptamer complexes into "open" and "closed" configurations [204]. The tweezer in the closed state I is composed of two arms (15) and (16) interlocked by two bridging strands, (17) and (18), that hybridize with counter complementary domains associated with the arms (15) and (16). The arms (15) and (16) include sequence-specific domains corresponding to the adenosine monophosphate aptamer, AMP aptamer. In the presence of AMP, the formation of the AMP ligand/AMP aptamer complexes releases the strand (18), resulting in the formation of the open tweezer configuration, state II. Subjecting the open tweezer to the enzyme adenosine deaminase transforms the AMP ligand to inosine monophosphate (IMP) that lacks affinity towards the AMP aptamer. This results in the rehybridization of the free strand (18) with the open tweezer to regenerate the closed tweezer state, state I. By the labeling of the inner interbridging strand (17) with a fluorophore-quencher pair, the dynamic opening and closure of the tweezers is followed spectroscopically. In the closed state of the tweezer, state I, the spatial proximity between the fluorophore-quencher units led to the effective quenching of the fluorophore. In turn, the AMP-guided separation of the tweezers through the formation of AMP/AMP aptamer complexes, state II, results in the spatial separation between the fluorophore-quencher units and the recovery of the fluorescence of the fluorophoreprobe. By the cyclic treatment of the tweezer with AMP and adenosine deaminase, the dynamic mechanical switching of the tweezer between the closed and open states was demonstrated, Figure 15B. The assembly of origami nanostructures by the programmed interaction of a long single-strand DNA, e.g., M13 phage, with hundreds of computer-designed "staple" oligonucleotide strands to yield 2D or 3D shapes has become a common practice in DNA nanotechnology. By the conjugation of nucleic acid tethers to the staple units comprising the origami scaffolds or the edges of the origami tiles, functional units for the successive modification of the origami structures were demonstrated [205,206]. Programmed attachment of nanoparticles [207,208], proteins [209] or polymers [210,211] to the protruding tethers associated with the origami structures was realized. These versatile functionalities of DNA origami nanostructures were applied to develop nanostructured aptamer-based origami switches and machines [212][213][214][215]. Figure 16A depicts the separation of two interlinked origami frames T 1 and T 2 through the formation of aptamer-ligand complexes [216]. The bridging of the origami frames by L 1 /L 1 duplex, where L 1 includes the aptamer sequence against ATP, provided the binding motif to assemble the dimer origami structure. In the presence of ATP, the formation of the ATP/ATP aptamer complexes led to the separation of the dimers. The dimer to monomer transitions were followed by atomic force microscopy, AFM ( Figure 16B). In addition, edge-crosslinked origami frames trimers, T 1 -T 3 -T 4 , were designed by the programmed modification of counter edges of the origami tile T 3 with the strands L 1 and L 2 and the subsequent hybridization of the L 1 -modified tile T 1 and of the L 2 -functionalized tile T 4 to yield the respective duplex bridged origami trimer T 1 -T 3 -T 4 . The strands L 1 and L 2 correspond to the ATP and the cocaine aptamer, respectively ( Figure 17A) [216]. In the presence of ATP, the origami frame could be separated, leading to the monomer T 1 and the dimer T 3 -T 4 . In the presence of cocaine, the trimer structure could be separated through the formation of the cocaine/cocaine aptamer complexes, leading to the formation of the T 1 -T 3 dimer and the T 4 monomer. In the presence of ATP and cocaine the trimer structure could be separated into individual origami frames, T 1 , T 3 and T 4 through the formation of the respective ATP/ATP aptamer complexes and cocaine/cocaine aptamer complexes. The specific frame structures could be identified by labelling of the frame T 1 with two nucleic acid hairpin markers (presented as dots) and labelling of the frame T 3 with a single hairpin marker (presented as a dot). The programmed dictated separation of the origami trimer by ATP, cocaine, and a mixture of ATP and cocaine could then be imaged by AFM and the quantitative analysis of the respective monomer/dimer compositions in the respective scan areas was also accomplished ( Figure 17B). Treatment of the origami trimer T 1 -T 3 -T 4 with ATP yield the separation of tile T 1 through the formation of the ATP/ATP aptamer complex and the intact T 3 -T 4 dimer. Subjecting the trimer to cocaine yield the T 1-T 3 dimer and separated tile T 4 through the formation of the cocaine/cocaine aptamer complex. Treatment of the trimer with cocaine and ATP led to the concomitant separation of the trimer to the monomer tiles T 1 , T 3 , T 4 . (B) AFM images corresponding to: Panel I-the origami trimer T 1 -T 3 -T 4 ; Panel II-the ATP triggered separation of the trimer T 1 -T 3 -T 4 into the monomer tile T 1 and the dimer tile T 3 -T 4 ; Panel III-the cocaine triggered separation of the trimer T 1 -T 3 -T 4 into the dimer tile T 1 -T 3 and the monomer tile T 4 ; Panel IV-the cocaine and ATP triggered separation of the trimer T 1 -T 3 -T 4 into the monomer tiles T 1 , T 3 , T 4 . The AFMimages are accompanied by statistical analysis of the percentage of the tiles. Reproduced with permission from ref. [216]. Copyright 2017 Royal Society of Chemistry.
A nanoengineered DNA-origami raft was designed as a functional nanostructure for the "mechanical" switchable opening and closure of a "window" in the raft ( Figure 18A) [217]. The origami tile was engineered to include a subdomain (window) linked to the raft by eight hinges, two hairpin "arms", H a and H b , and two duplex locks M/M' composed of two complementary strands M and M', where M includes the ATP sequence. In addition, the origami scaffold includes two additional protruding tethers, A 1 and A 2 , acting as capturing strands. In the presence of ATP and the added two hairpin structures, H 1 and H 2 , the two duplexes M/M' are unlocked and the opening of the hairpins H 1 and H 2 by hybridization with the arms H a and H b yield "ropes" that pool and open the window in the origami raft through hybridization ("binding") of the ropes with the anchor strands A 1 and A 2 . The reverse release of the "helper" rope strand with the strands H 1a ', H 1b ', H 2a ' and H 2b ' and the release of the ATP ligand by the counter ATP a ' aptamer strand, followed by washing of the resulting ATP-ATP a ' aptamer complexes, uncage the strands M and M' allowing the regeneration of the locked "window" origami. The unlocking of the closed origami tile configuration and the ATP-driven open "window" configuration of the nanocavity were imaged by AFM microscopy. The resulting AFM images, and the respective cross-section analyses of the respective tiles, are shown in Figure 18B,C. The reversible mechanical opening and closure of the cavities is demonstrated in Figure 18D. mechanical" open of a "window patch" in an origami tile using the formation of ATP/APT aptamer complexes as a mechanism to unlock the "window". The formation of the ATP/ATP aptamer complexes followed by the binding of helper strands to "window" handles and their stretching to bind the stature foothold, associated with the origami scaffold led to the fixed rigid opening of the "window" and to the formation of a nanocavity in the origami raft. The reverse unlocking of the foothold binding strand and the release of ATP relocks the caped "window" structure. (B) AFM images of the locked origami structures and accompanying cross-section analysis. (C) AFM image of the cavity containing unlocked origami rafts and accompanying cross-section analysis. (D) Switchable and reversible ATP-driven opening and closure of nanocavities in the origami raft. Reproduced with permission from ref. [217]. Copyright 2020 Royal Society of Chemistry.
In addition to the mechanical unlocking and closure of the nanocavities in the origami tiles, switchable "ON" and "OFF" catalysis in the confined nanoholes generated in the origami tiles was demonstrated. Towards this goal, the origami tile at the boundary of the "window" domain was functionalized on the upper face with two protruding tethers, T 1 and T 3 , and from the bottom face with two additional protruding tethers, T 2 and T 4 . The strand E 1a , E 1b , being subunits of the Mg 2+ -ion-dependent DNAzyme, were hybridized to the tethers T 1 /T 3 and T 2 /T 4 on the opposite faces of the origami tile. In the locked configuration of the tiles, the communication between the two subunits on the opposite faces of the origami tile is prohibited. The mechanical unlocking of the "window"allowed, however, the assembly of two Mg 2+ -ion-dependent DNAzyme structures in the confined cavity of the nanostructure. The hydrolytic catalyzed cleavage of the ROX/BHQ-functionalized ribonucleobase substrate of the DNAzyme units, resulted in the ROX fluorescence of the fragmented product generated by the supramolecular DNAzyme units (Figure 19). By the cyclic opening and closure of the "window" upon treatment of the system with ATP and its relocking by removal of the ATP, the catalytic process in the cavities was switched reversibly across "ON" and "OFF" states, Figure 19B. Figure 19. (A) ATP-driven opening and closure of a "window" in an origami scaffold and the triggered "ON"/"OFF" activation of the Mg 2+ -ion-dependent DNAzyme in the structurally confined cavity. Two subunits of the DNAzyme are positioned at the top and below the origami scaffold. Opening of the "window" leads to the self-assembly of the Mg 2+ion-dependent DNAzyme in the cavity resulting in the activation of the DNAzyme and the cleavage of the fluorophore/quenchermodified substrate. The fluorescence of the fragmented product transduces the activity of the DNAzyme. The closure of the "window" separates the two DNAzyme subunits leading to the switched off state of the DNAzyme. (B) Cyclic and switchable activation of the Mg 2+ -ion dependent DNAzyme in the cavity upon the switchable opening/closure of "window". Reproduced with permission from ref. [217]. Copyright 2020 Royal Society of Chemistry.

Conclusions
The specific recognition properties of aptamers have found broad applications in chemistry, medicine and materials science. Numerous applications of aptamers in analytical chemistry, e.g., sensing and separation, biomedical applications such as imaging, therapeutics and tissue targeting and material science, reflected by the development of new catalysts and the synthesis of soft materials, have been demonstrated in the past two decades. The integration of aptamers with nanoparticles, or the integration of aptamers with DNA or proteins, introduced important aptamer-nanoparticle hybrids and aptamer nanostructures, thus advancing the field of nanotechnology and nanobiotechnology. These aptamer-nanoparticle hybrids, or programmed aptamer nanostructures, combine the unique selective recognition and targeting properties of the aptamers with unique optical, electronic or catalytic properties of nanoparticles, or enabled the integration of aptamers with soft nanostructures, such as DNA tetrahedra or DNA origami, to yield functional materials for broad utility in the fields of sensing, imaging, therapy, drug delivery and more. The present perspective introduced some activities originated from our laboratory, demonstrating the applications of aptamer-functionalized hybrid structures for sensing, drug delivery, catalysis and mechanical features. Despite the progress in the field, one might identify future challenges. (i) Recent activities demonstrated the significance of molecular dynamic simulations to understand, and improve by base mutations, the ligand-aptamer interactions. Such molecular simulations followed by experimental validation could introduce superior aptamers. (ii)The chemical modification of aptamers by redox or photochemical functionalities led to aptamers of switchable binding properties and even enhanced binding properties. The introduction of new chemical functions into aptamer scaffolds is anticipated to lead to new applications of aptamers for sensing and molecular machine design. (iii) The functionalization of nanoparticles and nanomaterials with aptamers has been applied, until now, mainly to develop carriers for drug delivery and imaging. The recent extensive efforts to apply nanoparticles as catalytic "nanozymes" suggest that the functionalization of these particles with aptamers could yield improved "aptonanozymes", i.e., catalytic hybrids that concentrate the reaction substrate at the catalytic site, in analogy to native enzymes. In summary, the broad applications of aptamers demonstrated in the past two decades promise exciting future developments in chemistry, nanomedicine and material science.

Conflicts of Interest:
The authors declare no conflict of interest.