Aptamer‐Modified Homogeneous Catalysts, Heterogenous Nanoparticle Catalysts, and Photocatalysts: Functional “Nucleoapzymes”, “Aptananozymes”, and “Photoaptazymes”

Conjugation of aptamers to homogeneous catalysts (“nucleoapzymes”), heterogeneous nanoparticle catalysts (“aptananozymes”), and photocatalysts (“photoaptazymes”) yields superior catalytic/photocatalytic hybrid nanostructures emulating functions of native enzymes and photosystems. The concentration of the substrate in proximity to the catalytic sites (“molarity effect”) or spatial concentration of electron‐acceptor units in spatial proximity to the photosensitizers, by aptamer–ligand complexes, leads to enhanced catalytic/photocatalytic efficacies of the hybrid nanostructures. This is exemplified by sets of “nucleoapzymes” composed of aptamers conjugated to the hemin/G‐quadruplex DNAzymes or metal–ligand complexes as catalysts, catalyzing the oxidation of dopamine to aminochrome, oxygen‐insertion into the Ar─H moiety of tyrosinamide and the subsequent oxidation of the catechol product into aminochrome, or the hydrolysis of esters or ATP. Also, aptananozymes consisting of aptamers conjugated to Cu2+‐ or Ce4+‐ion‐modified C‐dots or polyadenine‐stabilized Au nanoparticles acting as catalysts oxidizing dopamine or operating bioreactor biocatalytic cascades, are demonstrated. In addition, aptamers conjugated to the Ru(II)–tris‐bipyridine photosensitizer or the Zn(II) protoporphyrin IX photosensitizer provide supramolecular photoaptazyme assemblies emulating native photosynthetic reaction centers. Effective photoinduced electron transfer followed by the catalyzed synthesis of NADPH or the evolution of H2 is demonstrated by the photosystems. Structure–function relationships dictate the catalytic and photocatalytic efficacies of the systems.


Introduction
Emulating the catalytic functions of native enzymes by artificial means is one of the long-term efforts in chemistry.Whilst homogenous and heterogenous catalysts play key roles in chemical catalysis, their ability to mimic the catalytic efficacies of native enzymes is still limited.Despite the diversity of chemical transformations driven by these catalysts, their operation usually requires high temperatures and operating in non-aqueous environments.Moreover, they exhibit limited selectivity and chiroselectivity and modest turn-over catalytic rates, far below the functions of native enzymes.[8][9] These included the assembly of catalyst-receptor conjugates, for example, catalyst-macrocyclic supramolecular structures [10] or catalyst-cyclodextrin conjugates, [11][12][13] catalyst-imprinted polymer matrices [14,15] or catalyst-imprinted nanoparticle aggregates. [16]While interesting artificial enzyme functions, such as stereoselectivity, chiroselectivity, and catalytic rate enhancement, were demonstrated by these systems, the enzyme-like catalytic performance of these systems and the practical applications of these artificial enzymes are limited.
[45] The aptamers are elicited via the systematic evolution of ligands by exponential enrichment (SELEX) procedures or related selection processes. [46,47]Chemical modification of aptamers with redoxactive [48] or photoactive constituents [49] led to redox-or photostimulated switching of aptamer binding functions.The selective binding properties of aptamers find broad applications in the development of sensor devices, [50][51][52] and medical applications, such as imaging [45,[53][54][55] or targeting of drug carriers to cells, [56][57][58][59] and therapeutic applications based on the inhibition of specific enzymes. [60,61]he diverse means to modify the 3′-or 5′-ends or internal positions of aptamer strands with catalytic DNAzymes or photocatalytic constituents allows the coupling of the unique binding properties of aptamers to catalytic/photocatalytic functionalities, yielding supramolecular assemblies mimicking native catalytic and photocatalytic systems.Herein, we highlight recent advances to tailor DNAzyme/aptamer, homogeneous catalyst/aptamer, heterogenous catalyst/aptamer and photocatalyst/aptamer conjugates as a versatile approach to mimic enzymes and photosynthetic systems.The possible applications and future perspectives of the field are discussed.
Figure 1 outlines the concept of the systems where a catalytic or photocatalytic site is conjugated to aptamer constituents.The conjugated aptamer provides a binding site for the reaction substrate in proximity to the catalytic site, emulating the catalytic and binding (molarity effect) features of the active site of native enzymes.In the photoaptazyme construct, a homogeneous photosensitizer or heterogeneous photosensitizer is conjugated to the aptamer site that binds an electron-acceptor ligand.The intimate contact between the photosensitizer and acceptor units provides a supramolecular structure for effective electron-transfer quenching and the activation of photoelectrocatalytic cascades.The different modes to conjugate catalyst/photocatalyst units to the 3′or 5′-ends of the aptamer, and the feasibility to link the aptamer sequences to the catalyst/photocatalyst, by spacer bridging tethers of variable lengths and shapes, allow then versatile means to synthesize diverse catalytic/photocatalytic hybrids enabling the evaluation of structure-function relationships and the resulting biomimetic efficacies of the systems.
Realizing that catalytic nanoparticles were termed "nanozymes" and that catalytic nucleic acids were defined as "DNAzymes", the enzyme-mimetic hybrid nanostructures consisting of catalytic nucleic acids and aptamer binding sites are termed by us as "nucleoapzymes" and the heterogenous catalytic nanoparticles coupled to aptamer binding constituents are defined as "aptananozymes".Similarly, the hybrid structures consisting of photosensitizer units linked to aptamer binding sites represent a biomimetic photocatalytic supramolecular structure defined by us as a "photoaptazyme".
Sections 2 and 3 of this account address the concept of nucleoapzymes, where the catalytic sites (DNAzymes or homogeneous transition metal catalyst) are conjugated to the aptamer substrate binding sites, resulting in supramolecular complexes exhibiting spatial proximity between the catalytic sites and binding sites as biomimetic models mimicking active site structures of native enzymes.The discussion introduces diverse examples  [68] Copyright 2016, American Chemical Society.
of catalyst/aptamer conjugates as a comprehensive framework aiming to emphasize the versatility of the concept of modeling enzyme functions.In particular, the structural modularity of the catalyst-aptamer conjugates (directionality of aptamer linkage or presence of spacer bridges) allows the construction of a rich library of structural hybrids that enable the identification of structure-catalytic function relationships, and eventually computational simulations to rationalize the catalytic behavior of the systems.

DNAzyme-Aptamer Conjugates as Enzyme Mimics: "Nucleoapzymes"
The nucleoapzyme concept is exemplified in Figure 2 with the synthesis of the hemin/G-quadruplex-dopamine binding aptamer (DBA) conjugates as dopamine oxidation nucleoapzymes catalyzing the H 2 O 2 -promoted oxidation of dopamine to aminochrome. [62]The nucleoapzymes consist of a hemin/Gquadruplex complex linked to the 5′-end of the aptamer through a single A-base bridge, configuration I, hemin/G-quadruplex bridged to the 5′-end of the aptamer through a longer bridging tether (TATA), configuration II, and the hemin/G-quadruplex linked to the 3′-end of the dopamine aptamer, configuration III (Figure 2A).Also, control systems, probing the oxidation of the dopamine by H 2 O 2 by the separated hemin/G-quadruplex and dopamine aptamer constituents, configuration IV, or the hemin/G-quadruplex conjugated to a scrambled nucleic acid sequence comprising the bases of the dopamine aptamer, configuration V, were evaluated.
The oxidation rates of variable concentrations of dopamine by H 2 O 2 , using the library of nucleoapzymes (Figure 2B) follow the order II > I >> III.All nucleoapzymes revealed a substantially enhanced catalyzed oxidation of dopamine as compared to the catalyzed oxidation of dopamine by the separated constituents or the randomized aptamer conjugated hemin/G-quadruplex, and all hemin/G-quadruplex-aptamer hybrids revealed Michaelis-Menten-type saturation kinetics, consistent with the saturation of the aptamer binding site with the dopamine substrate.
Whilst the results reveal that the association of the dopamine substrate to the aptamer receptor and the concentration of the substrate in proximity to the catalytic hemin/G-quadruplex mimic the active site functions of the native enzyme, significant structure-function relationships in the catalytic efficacies are observed.Tethering of the catalytic site to the 5′-end of the aptamer, configurations I and II, leads to effective nucleoapzymes (V max = 9.6 ± 0.5 nm s −1 and 13.5 ± 0.5 nm s −1 , k cat = 13 ± 0.7 × 10 −3 s −1 and 18.3 ± 0.9 × 10 −3 s −1 for I and II, respectively), yet the catalytic activity of the nucleoapzyme in configuration III is substantially lower.As the binding affinities of dopamine to all nucleoapzymes I-III were similar, it was concluded that although the concentration effect of all hybrid nucleoapzymes is identical, the spatial orientation of the substrate within the catalyst/aptamer complex plays an important role in determining activities of the catalysts.Indeed, molecular dynamics simulations (Figure 2C), demonstrated that in nucleoapzyme II the hemin/G-quadruplex catalytic unit is positioned in proximity (3-5 nm) with respect to the dopamine binding pocket, panel I, whereas in the least active nucleoapzyme III the catalytic site is positioned in a spatially remote structure with respect to the dopamine binding site, (9-15 nm), where the accessibility of the catalyst to the bound substrate is hindered, panel II.The enhanced catalytic activity of nucleoapzyme II as compared to I was attributed to the TATA bridge linking the aptamer to the hemin/G-quadruplex that enhanced the structural flexibility of the nucleoapzyme II structure, resulting in a closer spatial proximity between the catalytic site and the aptamer binding domain (3-5 nm vs 3-7 nm).
The concept of nucleoapzymes has been further extended to include a series of nucleoapzymes consisting of the hemin/Gquadruplex conjugated to arginine aptamers for the enhanced H 2 O 2 -driven oxidation of N-hydroxyarginine to citrulline, as compared to the oxidation of the substrate by the separated hemin/Gquadruplex and aptamer constituents.Whilst these systems validated the role of substrate concentration by the catalyst-aptamer conjugates to yield effective nucleic acid-based catalysts, the study emphasized the limitations of the approach rest on the scarce identification of DNAzyme/aptamer pairs to construct the nucleoapzyme hybrids for diverse chemical transformations.

Homogeneous Catalysts Conjugated to Aptamers as Functional Nucleoapzymes
A possible resolution to the limited variability of DNAzymeaptamer pairs to construct nucleoapzymes for diverse chemical transformations was introduced by substituting the DNAzyme catalyst units with homogeneous catalyst complexes coupled to the aptamer substrate binding constituents.According to this approach, the plethora of transition metal complexes acting as homogeneous catalysts could be coupled to the aptamer receptor ligands yielding a versatile concept to design novel watersoluble nucleoapzymes.The significance of the approach rests on the integration of DNA nanotechnology tools and catalysts to yield supramolecular, water-soluble, motifs consisting of transition metal complex-aptamer conjugates emulating the active site structure of native enzymes.
Figure 3 summarizes three examples of homogeneous catalyst-aptamer nucleoapzyme systems.Figure 3A depicts a set of Cu 2+ -terpyridine-functionalized DBA conjugates for the catalyzed H 2 O 2 oxidation of dopamine to aminochrome. [63]The set of nucleoapzymes consists of the Cu 2+ -terpyridine complexes linked directly to the 5′-end or 3′-end of DBA, configurations I and II, or nucleoapzymes conjugated to the 5′-end or 3′-end of DBA through a 4 × T spacer, configurations III and IV, respectively.
The catalytic performance of the nucleoapzymes was compared to the catalytic activity of the separated Cu 2+ -ion terpyridine and DBA constituent (Figure 3A).Whilst the Cu 2+ -terpyridinemodified DBA, configuration III, reveals the highest enzyme-like catalytic activities, k cat = 4 × 10 −3 s −1 , and demonstrates a 60fold enhanced oxidation rate as compared to the separated catalyst and aptamer constituents (Figure 3B, curve (a) versus curve (f)), the nucleoapzymes composed of the Cu 2+ -terpyridine linked directly to the 5′-end of the aptamer, configuration I, curve (b), the catalyst bound to the 3′-end of the aptamer through a 4 × T spacers, configuration IV, curve (c), or the catalyst conjugated to the 3′-end of the DBA, configuration II, curve (d), reveal comparable catalytic activities, V max = 25 ± 1 nm s −1 and k cat = 2.5 × 10 −3 s −1 values.As the binding constants of the dopamine substrate to the different nucleoapzymes were similar, the enhanced catalytic performance of nucleoapzyme I, as compared to the other supramolecular hybrids, was attributed to superior spatial interactions between the catalytic site and the substrate binding pocket.Indeed, molecular dynamics simulations suggested that the distance separating the Cu 2+ -terpyridine catalytic site and the binding site is ≈24 Å while the spatial separation of the active site from the binding site for the other nucleoapzymes is ≈30-32 Å.
Interestingly, a control system consisting of a Cu 2+terpyridine complex linked to a scrambled base-sequence of the DBA revealed substantially lower catalytic performance, as compared to the nucleoapzymes (Figure 3B, curve (e)), yet an apparent 14-fold catalytic enhancement, as compared to the separated constituents was observed.This rate enhancement was attributed to the non-specific electrostatic attraction of the positively charged dopamine to the negatively charged scrambled base aptamer strand, resulting in the non-specific concentration of the substrate in proximity to the catalyst.Related nucleoapzymes consisting of the Fe 3+ -terpyridine complexes linked to the 5′-end or 3′-end of the DBA through 4 × T spacers were reported (see Figure 3C), configurations X and XI, respectively.The nucleoapzyme X and XI revealed a 140-fold and 95-fold rate enhancement toward the oxidation of dopamine by H 2 O 2 to form aminochrome, as compared to the separated constituents.(V max = 26.7 ± 0.2 × nm s −1 and k cat = 2.67 × 10 −2 s −1 for X, V max = 20 ± 0.3 nm s −1 and k cat = 2.0 × 10 −2 s −1 for XI).
Hydrolytic transformations driven by transition metal complex-aptamer conjugates are exemplified in Figure 3D with the development of nucleoapzymes catalyzing the hydrolysis of ATP to ADP. [64] Realizing that many native phosphatases include in their active site Zn 2+ -ions as a co-factor, [65,66] and reports demonstrating the significance of dicationic activation (Zn 2+ ) of the phosphodiester linkage of ATP toward hydrolysis, [67][68][69] a series of bis-Zn 2+ -pyridyl-salen complexes conjugated to the ATP-aptamer were introduced as nucleoapzyme catalysts for hydrolysis of ATP (Figure 3D).The set of nucleoapzymes included the catalytic units directly linked to the 3′-and 5′-ends of the ATP-aptamer, configuration I and II, or linkage of the catalyst to the 3′-and 5′-ends of the ATP-aptamer through 2 × T spacer units, configuration III and IV.While the separated catalyst and aptamer constituents did not show any detectable hydrolytic activity, the nucleoapzymes demonstrated catalytic activities toward the hydrolysis of ATP to ADP (Figure 3E), following the order III (V max = 4.4 ± 0.5 μm min −1 , k cat = 6.88 min −1 ) > I (V max = 2.8 ± 0.4 μm min −1 , k cat = 4.37 min −1 ) > IV (V max = 1.9 ± 0.1 μm min −1 , k cat = 29.7 min −1 ) >> II (V max = 0.5 ± 0.1 μm min −1 , k cat = 0.78 min −1 ).Whilst the introduction of the 2 × T spacer bridges improved the catalytic activity of the nucleoapzymes, presumably due to spatial flexibility introduced between the active site and the ATP binding site, the positioning of the Zn 2+ -ion salen catalyst at the 3′-ends of the aptamer enhanced the hydrolytic activities of the nucleoapzymes as compared to the 5′-ended linkage of the catalyst to the aptamer.As all nucleoapzymes (I-IV) revealed similar binding affinities toward ATP (K d = 32 ± 1 μm), the differences in the catalytic activities of the hybrid catalyst/aptamer complexes were attributed to structural features of the resulting nucleoapzymes.Indeed, molecular  , d) II, e) the Cu 2+ -terpyridine complex linked to a scrambled DBA, f) separated Cu 2+ -terpyridine complex and DBA.C) A set of Fe 3+terpyridine DBA catalyzing the oxidation of dopamine to aminochrome, in the presence of H 2 O 2 .A-C) Reproduced with permission. [69]Copyright 2018, American Chemical Society.D) A set of bis-Zn 2+ -pyridiyl-salen-type complexes conjugated to ATP aptamers, acting as nucleoapzyme, catalyzing the hydrolysis of ATP to ADP. e) Rates of hydrolysis of ATP in the presence of different concentrations of ATP using the bis-Zn 2+ -pyridiyl-salen-type complex in the configuration: a) III, b) I, c) IV, d) II, e) V. D-F) Reproduced with permission. [70]Copyright 2020, Wiley-VCH.
dynamic simulations suggested that the distances separating the catalyst from the ATP binding site are substantially shorter in nucleoapzyme II as compared to I (Figure 3F).
Besides metal-ion co-factors playing important roles in activating esters toward hydrolytic transformations, amino acid residues associated with enzymes, such as lysine or histidine, provide general-base functionalities emulating the ester or amide hydrolytic transformations driven by native esterases or amidases.Previous studies demonstrated that the functionalization of synthetic receptors, such as crown ethers [70] or cyclodextrins [71] yield artificial enzyme assemblies.Accordingly, the functionalization of an aptamer receptor with the imidazole ligand provided  A,B) Reproduced with permission. [79]opyright 2018, Royal Society of Chemistry.C) Schematic stepwise oxygen insertion into the Ar-H bond of tyrosinamide and the subsequent oxidation of the catechol product into amidodopachrome, in the presence of a set of nucleoapzymes consisting of Fe 3+ -terpyridine-functionalized tyrosinamideaptamer nucleoapzyme conjugated XX-XXIII, in the presence of H 2 O 2 and ascorbic acid.D) Rates of tyrosinamide oxidation in the presence of variable concentrations of the tyrosinamide substrate using the nucleoapzymes in configurations XX-XXIII and control systems corresponding to the Fe 3+catalyst conjugated to the scrambled bare sequence of the tyrosinamide-aptamer, configuration V, and the separated Fe 3+ -terpyridine catalyst and the tyrosinamide-aptamer. e) ESR spectrum corresponding to the ascorbate radical and hydroxyl radical generated by treatment of the H 2 O 2 and ascorbic acid mixture with the Fe 3+ -terpyridine/tyrosinamide-aptamer conjugate, and suggested mechanism for the oxygen-insertion into the Ar-H bond.C-E) Reproduced with permission. [80]Copyright 2019, Wiley-VCH.
a means to develop an esterase-like nucleoapzyme.This is exemplified in Figure 4A with the modification of the cholic acid recognition aptamer with the imidazole ligand. [72]The imidazolefunctionalized aptamer acted as a nucleoapzyme for the hydrolysis of the coumarin-modified cholic acid ester (XII) (Figure 4B).The nucleoapzyme demonstrated Michaelis-Menten kinetics toward the ester substrate (k cat = 0.8 ± 0.1 h −1 ), and a 100-fold enhanced hydrolytic activity as compared to the separated imida-zole/aptamer constituents, consistent with the concentration of the reactive substrate in spatial proximity to the catalytic functionality.
Beyond the oxidative and hydrolytic transformations driven by nucleoapzymes, effective nucleoapzymes catalyzing oxygeninsertion reactions into Ar-H bonds were accomplished.This is exemplified in Figure 4C with the design of Cu 2+terpyridine-or Fe 3+ -terpyridine-modified tyrosinamide-aptamer nucleoapzymes catalyzing the oxygen insertion process into the Ar─H moiety of tyrosinamide to yield the respective catechol product using H 2 O 2 as the oxygen source, in the presence of ascorbate as a co-factor. [73]The resulting amidated L-DOPA product was, then, oxidized by H 2 O 2 , in the presence of the nucleoapzymes, into amidodopachrome.
A series of nucleoapzymes composed of the catalysts conjugated to the 3′-ends or 5′-ends of the tyrosinamide-aptamer directly, configurations XX and XXI, or through 4 × T spacer bridges, configurations XXII and XXIII, were synthesized (Figure 4C), and the kinetic results corresponding to the stepwise oxidation of tyrosinamide to amidodopachrome, by the set of Fe 3+terpyridine-aptamer nucleoapzyme conjugates, are displayed in Figure 4D.
All nucleoapzymes revealed Michaelis-Menten saturation kinetics, demonstrating substantially enhanced reaction rates, as compared to the separated catalyst/aptamer constituents, following the order XXII > XX > XXIII > XXI.The most effective nucleoapzyme composed of configuration XXII demonstrated a ≈120-fold rate enhancement as compared to the separated constituents.As the binding affinities of the reaction substrate to the aptamer receptors were similar (K d = 14 μm), the enhanced catalytic functions of nucleoapzyme XXII were attributed to the spatial proximity between the Fe 3+ -terpyridine catalytic site and the substrate binding site.Interestingly, the biocatalytic oxygen insertion process into the Ar-H bond required the co-participation of ascorbate in the H 2 O 2 in the reaction mixture.Electron spin resonance (ESR) experiments revealed that catalytic formation of ascorbate and hydroxyl radicals was observed in the presence of the two reactants and the Fe 3+ -terpyridine nucleoapzyme effective (Figure 4E).Accordingly, a feedback mechanism for the amplified generation of •OH by the nucleoapzymes and the cooperative participation of the ascorbate radical and of hydroxyl radical species in the Ar─H oxygen insertion and subsequent oxidation of the catechol product was suggested.

Heterogenous Nanoparticle-Aptamer Conjugates for Catalysis: "Aptananozymes"
In this section, we introduce heterogenous nanoparticle/aptamer conjugates, aptananozymes, as hybrid heterogenous nanostructures emulating the function of native enzymes.While heterogenous catalysts usually lack effective and selective binding sites, the conjugation of aptamers to the heterogenous catalytic interfaces adds a functional selective binding dimension to the hybrid catalyst emulating enzyme functions.The unique features of aptananozymes as compared to native enzymes should be, however, mentioned.The nanometer-sized dimension of the nanoparticles, exhibiting comparable sizes to enzymes, include numerous catalytic sites, as compared to a single active site enzymatic contract.Furthermore, the ability to link many aptamer strands to the nanoparticle core, yields a multi-functional nanoparticle composite consisting of a cluster of active-site-binding site constituents.While the availability of clustered active sites/binding sites may confer highly active biomimetic agents, issues such as deactivation of the active site by the surface-linked aptamer or perturbation of the binding affinities of the aptamers by the heterogenous surface may arise.The following discussion aims to address these issues upon developing this class of nanomaterials.[76][77] Inorganic nanoparticles (NPs), such as Au, [78][79][80] Pt, [81,82] Pd NPs, [83,84] inorganic metal oxides or sulfides, for example, Fe 3 O 4 , [85][86][87] V 2 O 5 , [88,89] CeO 2 [90] or NiCo 2 S 4 , [91] carbon-based nanomaterials, such as carbon dots (C-dots), [92] graphene sheets [93] or quantum dots, [94,95] metal-organic framework NPs, [96,97] and organic nanoparticles, such as melanin [98,99] or polydopamine, [100,101] represent examples of catalytic-enzyme-like catalysts.
Diverse catalytic transformations driven by nanozymes mimicking native enzymes, such as oxidase, [102][103][104][105] peroxidase, [106][107][108] superoxide dismutase, [109] and hydrolase [110] were reported.Different applications of nanozymes for sensing, [111][112][113][114] imaging [115][116][117] and biomedical uses, such as cancer therapeutics treatment [118][119][120] or for the treatment of other diseases, for example, Alzheimer's [121,122] or Parkinson's diseases, [123] and their use as antibacterial agents [124,125] and catalysts for degradation of environmental pollutants [126] were realized.The different methods to synthesize and scale the preparation of nanozymes, and their stabilities and cost-effectiveness render nanozymes as effective catalysts for practical applications.Nonetheless, their catalytic performance, as compared to native enzymes, is still low, mainly due to poor binding interactions to the catalytic interfaces.Thus, the development of means to concentrate the substrates at the nanozyme interface is essential for the improvement of these catalysts.Several approaches were suggested to achieve this goal, including the functionalization of the nanozymes with substrate-binding receptors, for example, -cyclodextrin, [92] or the modification of the nanozymes with molecularly imprinted coatings. [16]The functionalization of nanozymes with aptamer strands may, thus, provide a versatile approach to concentrate the substrate at the nanozyme catalytic interfaces yielding nucleic acid-functionalized catalytic nanoparticles emulating native enzymes, "aptananozymes".
Recent reports demonstrated the successful engineering of aptamer-functionalized nanozyme systems, [127] aptananozymes, and the results sparked interest in the potential practical applications of these systems.Figure 5A introduces aptamerfunctionalized/Cu 2+ -modified carbon dots (C-dots) as aptananozymes for the catalyzed oxidation of dopamine by H 2 O 2 to form aminochrome.The C-dots, prepared by microwave pyrolysis of citric acid and urea, include surface amine (NH 2 ) and carboxylic acid (COOH) functionalities, and these acted as ligands for the binding of Cu 2+ -ions.In addition, the aminomodified DBA was covalently linked to free carboxylic acid units, associated with the Cu 2+ -ion-modified C-dots (approximately four aptamer strands per C-dot).
A series of aptananozymes I-IV, comprising aptamers linked directly through the 5′-end (I) or 3′-end (II) to the Cu 2+ -functionalized C-dots or aptamer linked to the Cu 2+functionalized C-dots through spacer bridges of variable lengths, (TGTA), (TGTA) 2 , (TGTA) 3 , were explored (configurations III-V).All aptananozymes revealed, in the presence of H 2 O 2 , enhanced catalyzed oxidation rates of dopamine to aminochrome, as compared to the separated Cu 2+ -ions-modified C-dots and the aptamer constituents (Figure 5B).While the aptananozyme IV revealed a 50-fold rate enhancement for the oxidation of dopamine, the set of aptananozymes displayed obvious structure-function  A-F) Adapted with permission. [135]Copyright 2021, American Chemical Society.
relationships, revealing a catalytic activity order IV > III > I > II > V.
The composition of all aptananozymes was very similar (Cu 2+ion content and aptamer loading), and different structural and functional parameters were suggested to affect the relative activities of the aptananozymes: i) The lower activity of aptananozyme II as compared to I was attributed to the lower binding affinity of dopamine to the aptananozyme II as compared to I (K d = 0.98 ± 0.06 μm of I; K d = 3.6 ± 0.2 μm of II).ii) The 5′-ended aptamers linked to Cu 2+ -ion-modified C-dots revealed similar binding affinities toward the substrates, yet strong dependence on the length of the spacer units bridging the aptamer to the catalytic interface led to catalytic efficiency in the order IV ≈ III > I ≫ V.The superior activities of aptananozymes IV and III as compared to I were attributed to the flexibility of the dopamine/aptamer complex introduced by the spacer groups that facilitates spatial proximity between the bound substrate and the catalytic interface.The lowest catalytic activity of aptananozyme V, composed of a long spacer bridge (TGTA) 3 , was attributed, despite the flexibility of the tether, to the spatial separation of the substrate from the catalytic Cu 2+ -ion-functionalized C-dots interface.
A related Cu 2+ -ion-modified C-dots aptananozyme system catalyzing the H 2 O 2 -stimulated oxygen insertion into the Ar-H bond of tyrosinamide to yield the catechol product that is subsequently oxidized to amidodopachrome was demonstrated (Figure 5C).(This system provides the heterogeneous aptananozyme analog to the Fe 3+ -terpyridine-tyrosinamideaptamer nanozyme system, cf.Section 3).A set of aptananozymes consisting of the tyrosinamide-aptamer linked to Cu 2+ -ion-modified C-dots were synthesized (Figure 5C), where the aptamers were linked directly to the carboxylic-acidfunctionalized catalytic interface using amino-modified 3′-end or 5′-end of the aptamers or through TGTA spacer bridges of variable lengths, aptananozymes (X)-(XIV).All hybrid aptananozymes demonstrated enhanced catalytic activities toward the oxygen-insertion process into the tyrosinamide substrate and the subsequent oxidation of the catechol product to dopachrome, in the presence of the ascorbate/H 2 O 2 mixture, as compared to the separated nanoparticle and aptamer constituents (Figure 5D).
The catalytic performance of the aptananozymes demonstrated structure-function relationships and followed the order XIII > XII > X > XI > XIV, where the most effective aptananozyme IV revealed a 60-fold enhanced catalytic activity, as compared to the separated constituents.The lower activity of the aptananozyme XI as compared to X was attributed to the lower binding affinity of the 3′-end tethered aptananozyme, XI, toward the substrate (K d (XI) = 1.2 ± 0.5 μm, K d (X) = 0.83 ± 0.01 μm).In turn, the other 5′-ended aptananozymes revealed similar binding affinities toward the substrate, and the order of the catalytic activities was attributed to the flexibility of the aptamer units introduced by the spacer groups, allowing closer proximity of the bound substrate to the catalytic interface.The lowest activity of the aptananozyme XIV was attributed, however, to the spatial separation of the substrate-aptamer complex from the catalytic interface, introduced by the long (TGTA) 3 spacer bridge.
ESR experiments revealed the participation of the hydroperoxyl radicals (•OOH) and ascorbate radicals (•AA) as essential reactive intermediates to drive the oxygen insertion process into the Ar-H bond of the tyrosinamide substrate (Figure 5E).The tentative catalytic cycle shown in Figure 5F was suggested to account for the catalytic aptananozyme activities.
Aptamer-modified AuNPs hybrids acting as aptananozymes were synthesized and the nanoparticles demonstrated dual catalytic activities toward the H 2 O 2 -catalyzed oxidation of dopamine to aminochrome and oxidase-like catalytic activities driving the aerobic oxidation of glucose to gluconic acid and H 2 O 2 . [128]These features allowed the use of the dopamine-aptamer-modified Au nanoparticles as aptananozymes for the oxidation of dopamine, and as a bioreactor system driving the cascaded oxidation of dopamine to aminochrome, through the primary aerobic oxidation of glucose by the catalytic nanoparticles.
Figure 6A depicts schematically the structure of the polyadenine-dopamine-aptamer conjugate, DBA-pA stabilized AuNPs used as aptananozymes for the catalytic oxidation of dopamine to aminochrome, in the presence of H 2 O 2 .A series of aptananozymes I-V consisting of DBA pA conjugates linked to the AuNPs through the 3′-or 5′-ends (I and II, respectively) or through a spacer bridged 5′-end-DBA-aptamer, III-II, acted as aptananozymes (Figure 6B).All aptananozymes revealed enhanced catalytic performance toward the H 2 O 2 oxidation of dopamine to aminochrome as compared to the separated constituents, following the order IV > III > II > I >> V, where the aptananozyme IV revealed a 55-fold enhanced activity as compared to the separate pA-AuNPs/DBA constituents (Figure 6C).Also, the set of aptananozymes revealed structurefunction relationships, analogous to those found for dopamine aptamer/Cu 2+ -ion-C-dots aptananozyme series.The lower catalytic activity of I, as compared to II, was attributed to the lower binding affinity of dopamine to the aptamer conjugated to the pA-AuNPs interface through the 3′-end.The most effective aptananozyme IV was rationalized by the highest binding affinity of the aptamer toward dopamine (molarity effect) and the flexibility of the spacer group composed of (TGTA) 2 units.Interestingly, whilst surface modification of AuNPs is known to inhibit the catalytic activities of AuNPs toward the aerobic oxidation of glucose, the DBA-aptamer-pA functionalized AuNPs act as effective oxidase mimicking catalysts for the aerobic oxidation of glucose to yield gluconic acid and H 2 O 2 .
These dual catalytic functions of the DBA-pA AuNPs were used to apply the nanoparticles as a bioreactor aptananozyme systems driving the cascaded oxidation of dopamine to aminochrome, using the nanozyme-catalyzed aerobic oxidation of glucose as the source of H 2 O 2 (Figure 6D).The set of DBA-pA AuNPs aptananozymes I-V was employed to stimulate the biocatalytic cascade (Figure 6E).
All aptananozymes demonstrated enhanced biocatalytic cascade activities, as compared to the separated pA-AuNPs/DBA constituents.The catalytic activities of the bioreactor aptananozymes system followed the order IV > III > II > I > V, and the activity of the aptananozyme IV/bioreactor system revealed a ≈50-fold enhanced as compared to the separated pA-AuNP and DBA constituents.Whilst the lower activity of I, as compared to II, was attributed to the lower binding affinity of aptananozyme I, the similar binding affinities of the aptamer constituents in II, III, and IV suggested that the flexibility of the aptamer chains, and the resulting spatial proximity between the dopamine substrate and the catalytic interface, control the catalytic performance of the aptananozymes.
Mechanistic studies demonstrated that the pA-AuNPs hybrids catalyze the formation of •OH (hydroxyl radicals) in the presence of H 2 O 2 (or indirectly by H 2 O 2 generated by aerobic oxidation of glucose).The resulting •OH was suggested as the reactive ROS intermediate that oxidized dopamine.
The overexpressed concentrations of H 2 O 2 in cancer cells, and the high metabolic glucose concentrations in cancer cells, suggested that the pA-AuNPs could act as catalysts for the chemodynamic treatment of cancer cells.Toward this goal, polyadenine conjugated to the AS1411-aptamer sequence was used to stabilize the AuNPs (Figure 7A).
The AS1411-aptamer-functionalized pA-AuNPs hybrid exploited the specific binding properties of the AS1411-aptamer to the nucleolin receptors associated with cancer cells, [129,130] and the catalytic features of the pA-AuNPs to catalyze the generation of cytotoxic ROS agents (•OH) for harnessing the aptananozyme for the selective chemodynamic treatment of cancer cells.Indeed, effective and selective AS1411-aptamer-guided formation of •OH species in MDA-MB-231 breast cancer cells, as compared to epithelial non-cancerous MCF-10A breast cells, was demonstrated (see Figure 7B).
The efficient aptananozyme-catalyzed generation of the •OH was applied for selective in vitro treatment of MDA-MB-231 breast cancer cells and in vivo chemodynamic treatment of Rates of dopamine oxidation to aminochrome in the presence of variable dopamine concentrations using the aerobic oxidation of glucose, 50 mm, by the bioreactor aptananozyme systems I-V and respective control systems.A-E) Adapted with permission. [136]Copyright 2022, American Chemical Society.Whilst high growth rates of the tumors were observed in the presence of non-aptamer pA-AuNPs or base-randomized AS1411-aptamer functionalized pA-AuNPs, the growth rate of the MDA-MB-231 tumors treated with the AS1411-aptamer modified pA-AuNPs was significantly dampened and, after a time-interval of ≈23 days, the size of the tumors were almost  A-C) Adapted with permission. [136]Copyright 2022, American Chemical Society.

MDA-MB-231 tumors, in xenograft-tumor-bearing mice (
identical to the original values.No changes in the mice's body weights were observed during the experiment, implying a lack of cytotoxicity of the AS1411-aptamer pA-AuNPs.The selective permeation of the AS1411-aptamer/pA-AuNPs into the cancer cells and the effective chemodynamic intracellular generation of •OH as a toxic ROS agent led to the selective therapeutic treatment of the cancer tumors.

Photosensitizer/Aptamer-Electron-Acceptor Hybrids as Artificial Photosynthetic Model System: "Photoaptazymes"
[133] Besides the fundamental scientific challenge in emulating the functions of the photosynthetic apparatus, such systems are of practical interest as they provide means for the conversion of solar light energy to electrical power and, moreover, can eventually lead the photoinduced synthesis of fuels (H 2 or CO 2 -fixation products). [134,135]he uniqueness of the photosynthetic apparatus rests on the evolutionary, optimized structures of the photosystems comprising the photosynthetic apparatus that leads to the light harnessing of the photosystems by photosynthetic antenna constituents, and to effective photoinduced vectorial electron transfer that spatially separates the electron-transfer intermediates and prohibits destructive back-electron-transfer reactions. [136,137]he photogenerated redox species in the photosynthetic reaction centers (quantum yield ≈ 1) are, then, coupled to dark chemical reactions, resulting in the biocatalyzed formation of NADPH and its utilization in the CO 2 -fixation cycle (Calvin cycle), and the concomitant oxygen evolution process.
Diverse chemical methods were introduced to mimic photophysical steps and catalytic chemical events of native photosynthesis.Indeed, molecular, supramolecular nanomaterialbased assemblies, and organized microheterogeneous environments were introduced to stimulate effective photoinduced electron-transfer and vectorial electron-transfer chains leading to efficient charge separation and retardation of destructive back electron transfer.These included photosensitizer-electronacceptor triads, [138][139][140] nanomaterial-based systems such as Auphotosensitizer-electron-acceptor aggregates [141,142] or metalorganic framework nanoparticles, [143,144] and microheterogenous environments, such as micellar, [145] liposomes, [146] or colloidal nanoparticles. [147,148]n addition, chemical transformations driven by photogenerated redox species, in the presence of homogeneous, heterogenous, or enzyme catalysts were demonstrated.For example, homogenous Co(III) or Pt(IV) complexes were applied for photocatalyzed H 2 -evolution, [149][150][151][152] heterogeneous Pt, Pd, and Ru nanoparticles were used as catalysts for H 2 evolution [153][154][155][156] or CO 2 -fixation, [134] and enzymes were coupled to photogenerated redox species to catalyze the photocatalyzed H 2 -evolution [157,158] or the light-induced biocatalyzed generation of NADPH and cascaded synthesis of amino acids [159] or CO 2 -fixation products. [160]he successful conjugation of catalytic sites to aptamer constituents to yield effective supramolecular catalytic assemblies, cf.Sections 2 and 3, suggested that the conjugation of a photosensitizer unit to an aptamer receptor that binds an electron-acceptor constituent (Figure 8A), could provide the basic supramolecular structure that emulates the functions of a photosynthetic reaction center. [161]The intimate spatial distance between the photosensitizer and the electron acceptor, and the non-covalent interactions between the electron acceptor and the aptamer, allow rapid exchange of the electron-transfer product with the bulk solution constituents, providing means for effective photoinduced electron transfer and charge separation.The subsequent utilization of the separated electron-transfer product for dark photosynthetic transformations is, then, feasible.
The set of photoaptazymes demonstrated high photocatalytic efficacies toward the photosensitized reduction of TA-MV 2+ to the reduced product TA-MV + •, in the presence of Na 2 EDTA as sacrificial electron donor (Figure 8B).The yield of photosensitized reduction of TA-MV 2+ to TA-MV + • by the photoaptazymes was substantially higher as compared to the separated Ru(II)tris-bipyridine/TA-aptamer system or the Ru(II)-tris-bipyridinescrambled TA-aptamer conjugate, where only traces of TA-MV + • were generated (Figure 8C).
The photoaptazyme performance demonstrated structurefunction relationships following the order IV > III > II > I, where the photoaptazymes consisting of the photosensitizer linked through the aptamer to the 4 × T spacer bridge revealed a higher binding affinity toward the TA-MV 2+ electron acceptor (K d ≈ 90 ± 20 nm) as compared to the other photoaptazymes.The photocatalytic activities of the photoaptazymes are controlled by the electron-transfer quenching efficiencies within the respective supramolecular photosensitizer-aptamer/TA-MV 2+ complexes that followed the order IV (k q = 1.43 × 10 −8 s −1 ) > III (k q = 1.25 × 10 −8 s −1 ) > II (k q = 1.05 × 10 −8 s −1 ) > I (k q = 0.87 × 10 −8 s −1 ).The differences in the electron-transfer quenching rate constants (and the resulting differences in the photocatalytic generation of TA-MV + • by the different photoaptazymes) were attributed to the difference in the binding affinities of TA-MV 2+ to the photoaptazymes and to the structural flexibility introduced by the 4 × T spacer bridges that allow enhanced spatial proximity between the bound TA-MV 2+ and the photosensitizer.
The superior photocatalytic functions of the photoaptazymes as compared to the separated constituents are reflected in the light-induced photosynthetic transformations.Figure 8D depicts the cascaded ferredoxin-NADP + -reductase, FNR, catalyzed reduction of NADP + to NADPH by TA-MV + • generated by the set of photoaptazymes in comparison to the separated constituents.Figure 8E presents the TA-MV + • mediated H 2 -evolution in the presence of Pt NPs as catalyst driven by the photoaptazymes I-IV, in comparison to the separated photosensitizer/aptamer constituents.The efficiencies of the photocatalyzed formation of NADPH or the H 2 -evolution process follow the primary photocatalytic electron-transfer features of the photoaptazymes.The photosynthetic transformations guided by the photoaptazymes are substantially enhanced as compared to the separated constituents.For example, the quantum yield for the formation of NADPH by photoaptazymes IV corresponds to 4.1% whilst the quantum yield for the generation of NADPH by the separated constituents is ≤0.2%.Similarly, the H 2 -evolution quantum yield by photoaptazymes IV corresponds to Φ = 3.9%, whereas inefficient H 2 -evolution is observed in the presence of the separated constituents, Φ = 0.31%.A further photoaptazyme system emulating elements of the photosynthetic reaction center and consisting of an all-DNA photosensitizer-aptamer framework [162] is displayed in Figure 9.
The system made use of the fact that Zn(II) protoporphyrin IX, Zn(II)PPIX, binds to G-quadruplexes and the photophysical properties of the Zn(II)PPIX are improved (enhanced fluorescence quantum yields). [163]ccordingly, nucleic acid strands consisting of a G-quadruplex domain that binds the Zn(II)PPIX photosensitizer, conjugated to the tyrosinamide aptamer, TA-aptamer, were designed as photosystem mimicking scaffolds (Figure 9A).The photosystem scaffolds included the conjugation of the 3′-end or 5′-end of the TAaptamer to the Zn(II)PPIX/G-quadruplex photoactive units, configuration I and II, and the conjugation of the 3′-end or 5′-end of the TA-aptamer to the Zn(II)PPIX/G-quadruplex framework through 4 × T spacer units, configuration III and IV, respectively.The association of TA-MV + • to the aptamer binding sites yields the supramolecular photosensitizer/electron-acceptor complex, where spatial proximity between the photosensitizer and aptamer-bound TA-MV 2+ promotes effective electron transfer.A-E) Adapted with permission. [174]opyright 2019, Wiley-VCH.
The resulting non-covalently bound reduced photoproduct, TA-MV + •, is, then, exchanged with TA-MV 2+ units solubilized in the bulk solution.The separated TA-MV + • product is, then, coupled to secondary electron transfer cascaded "dark" chemical transformations (Figure 9B).The set of photosensitizeraptamer conjugates I-IV reveals substantially enhanced yields of TA-MV + •upon steady-state irradiation of the conjugates in the presence of TA-MV 2+ and 2-mercaptoethanol as a sacrificial electron donor, as compared to the control systems comprising the Zn(II)PPIX/G-quadruplex conjugated to nonbinding scrambled-base aptamer sequence or the separated Zn(II)PPIX/G-quadruplex and TA-aptamer constituents that demonstrated only trace yields of TA-MV + • under similar conditions (Figure 9C).
The effective photoinduced electron-transfer process was followed by effective charge separation and stabilization of the electron transfer against back reaction.The electron-transfer products Zn(II)PPIX + •/G-quadruplex and TA-MV + • demonstrated a long recombination time decay (1-1.5 ms) and a back reaction rate constant estimated to be k b ≈ 2-4 × 10 5 m −1 s −1 ).The slow back reaction rate was attributed to the steric barrier introduced by the G-quadruplex in which the Zn(II)PPIX + • is embedded and the electrostatic repulsion between the exchanged Zn(II)PPIX + •-G-quadruplex-aptamer/TA-MV 2+ complex and the separated TA-MV + • photoproduct.The superior photosensitized electron transfer in the supramolecular Zn(II)PPIX-G-quadruplex/TA-MV 2+ photosystem conjugates, and the stabilization of the redoxproducts against recombination, led to the efficient generation of TA-MV + • under steady-state illumination, in the presence of the sacrificial electron donor (Figure 9A).
The efficient formation of TA-MV + • under steady-state irradiation was then coupled to the secondary TA-MV + •mediated ferredoxin-NADP + -reductase, FNR-catalyzed reduction of NADP + to NADPH (Figure 9E).Whilst the control systems consisting of separated Zn(II)PPIX/G-quadruplex photosensitizer and TA aptamer assembly or the Zn(II)PPIX/Gquadruplex linked to the scrambled base-sequence of the TA aptamer revealed only trace efficacies toward the photosynthesis of NADPH (in the presence of TA-MV 2+ and the sacrificial electron donor and under steady-state illumination), all of the engineered photoaptazymes photosensitizer/aptamer photosystem conjugates displayed effective photosynthesis of NADPH.The rates of photosynthesis of NADPH by the photosystems were guided by the primary photoinduced electron-transfer features of the photosystems, III > IV > I > V (Figure 9F).
In addition, by applying the rolling circle amplification (RCA) nanotechnological tool, [50,164] the scaling of the photoaptazymes concept was accomplished.This is exemplified in Figure 9G with the synthesis of Zn(II)PPIX/G-quadruplex-TA aptamer biopolymer chains.A circular DNA template was engineered to include the promoter Z sequence, the sequence X complementary to the TA-aptamer and linked through a 4-adenine base spacer to the sequence Y, which is complementary to the G-quadruplex sequence, and was employed to stimulate the RCA process.In the presence of polymerase and a mixture of dNTPs, and co-added Zn(II)PPIX, the RCA process synthesized polymer chains consisting of repeat units composed of Zn(II)PPIX photosensitizer units linked to two conjugated TA-aptamer constituents, for example, panel I.As two TA-MV 2+ electron acceptors are adjacent to the photosensitizer, superior TA-MV 2+ electron-transfer quenching of Zn(II)PPIX/G-quadruplex sites, proceed in the polymer scaffold, as compared to the Zn(II)PPIX/G-quadruplex-TA aptamer conjugate.
Polymer chains of Zn(II)PPIX/G-quadruplex-TA aptamer conjugate exhibiting lengths corresponding to 700-800 nm were formed (Figure 9G, panel II).While the separated constituents revealed negligible photosynthesis of NADPH, the biopolymer showed an approximately threefold enhancement, as compared to the biaptamer/Zn(II)PPIX conjugate, curve (b), in the production of NADPH.This enhancement was attributed to the entanglement of the polymer chains and to the cooperative electrontransfer quenching of the photosensitizer units by entangled Zn(II)PPIX/G-quadruplex-TA-aptamer-TA-MV 2+ units, beyond the neighboring aptamer-TA-MV 2+ quencher units.Thus, the scaling of the photoaptazymes structure into a flexible polymer chain resulted in, the effective concentration-guided electron-transfer quenching process within the supramolecular Zn(II)PPIX/G-quadruplex-TA-MV 2+ complex composite, demonstrating a cooperative assisted electron-transfer quench induced by the entangled flexible photoaptazyme polymer chains.
While the "nucleoapzymes" and "aptananozymes" emulated the active-site functions of native enzymes by concentrating the reaction substrate (molarity effect) in spatial proximity to the catalytic sites, the "photoaptazymes" acted as functional scaffolds mimicking the photosynthetic reaction centers.Sets of different structures of "nucleoapzymes", "aptananozymes" and "photoaptazymes" were designed and characterized toward target reactions or chemical transformations.Structure-function relationships within the sets of catalysts/photocatalysts were recognized.
While all "nucleoapzymes", "aptananozymes" and "photoaptazymes" demonstrated enhanced catalytic or photocatalytic transformations as compared to the separated catalyst/photocatalyst and aptamer constituents, several parameters were found to control the catalytic/photocatalytic efficacies of the frameworks.
i) The binding modes of the aptamer to the catalyst/photocatalyst affect the binding affinities of the substrate/quencher toward the aptamer, thereby controlling the substrate/quencher concentration capacities of the systems and the resulting catalytic/photocatalytic efficacies of the hybrid nanostructures.The catalytic/photocatalytic units perturb the aptamer/ligand binding affinities, as compared to "bare" aptamer sequences.ii) The introduction of spacer bridging units between the aptamer and the catalyst/photocatalyst sites improved the catalytic/photocatalytic performance of the nucleoapzymes/aptananozymes/photoaptazymes.The introduction of the spacer groups improved the binding affinities of the substrates/electron-acceptor quenchers to the different aptamers, presumably by eliminating the perturbing effects of the catalytic/photosensitizer constituents on the binding affinities of the aptamer receptors.The enhanced spacer-stimulated binding affinities are, then, reflected in the catalytic/photocatalytic efficacies.iii) The length of the spacer units affects the catalytic performance of the nucleoapzymes/aptananozymes.Extending the lengths of aptamers with the spacer groups improved the catalytic performance of the nucleoapzymes/aptananozymes, presumably by enhancing the flexibility of the hybrid conjugates thereby facilitating the spatial proximity between the substrates and the catalytic site.Longer spacer units revealed, however, an adverse effect on the catalytic performance of the nucleoapzyme/aptananozyme, presumably due to the spatial separation between the catalytic site and the substrateaptamer complex.Thus, tuning and optimization of the length of spacer bridges in the catalytic conjugates were important.
To date, the parameters controlling the catalytic/photocatalytic functions of the hybrid nanostructures are merely qualitative and based on a small range of structural modifications within the hy-brid systems.A systematic quantitative assessment of functional parameters controlling the performance of the systems is certainly needed.This is, however, a scientific challenge since opposing effects such as the perturbation of the aptamer binding properties by the catalytic interfaces or the spatial disturbance between the catalytic site and the binding site at long separation distance might adversely affect the overall activities of the biomimetic enzyme models.Nonetheless, recent efforts [165] to computationally rationalize the binding properties of aptamers and catalytic properties of DNAzymes were reported.These computational tools could provide effective means for the future rational design of biomimetic enzyme model nanostructures.
Furthermore, a major advantage of synthetic nucleoapzymes and aptananozymes could involve the development of biomimetic enzyme models catalyzing non-native chemical transformations.The feasibility of eliciting aptamers for almost any target ligand by the SELEX procedure provides, then, means to anchor diverse aptamers to homogenous or heterogenous catalysts to yield nucleoapzymes or aptananozymes for a plethora of chemical processes.
An important issue related to the development of nucleoapzymes or aptananozymes rests on the potential future applications of these systems beyond chemical catalysis.The fact that aptamers act as selective high-affinity binding units toward ligands linked to catalytic agents yields hybrid systems for amplified selective and sensing applications.In fact, throughout the present report, many examples presented the development of nucleoapzymes or aptananozymes as superior catalysts for the oxidation of dopamine.The analysis of the dopamine neurotransmitter attracts substantial analytical interest, [166][167][168] and thus these nucleoapzymes/aptananozymes could provide sensing matrices for the neurotransmitter.
Furthermore, we emphasized the potential therapeutic applications of aptamer-modified nanozymes.The development of nucleoapzymes/aptananozymes combines the therapeutic and sensing efficiencies into integrated sense-and-treat systems that could provide versatile means for future nanomedicine.Indeed, in a recent report [169] the integration of catalytic metalorganic-framework nanoparticles and the hemin/G-quadruplex constituent was reported as an effective biocatalytic sense-andtreat bioreaction for the oxidation of N-hydroxy-L-arginine to citrulline and NO, an important agent for cardiovascular disorder applications.
While the results demonstrate the viability of the concept that exploiting the binding properties of nucleic acids can be utilized to develop effective new catalysts/photocatalysts emulating enzymes and photosynthetic reaction centers, the performance of the artificial catalysts and photocatalysts are still far from these of native systems.Thus, scientific pathways to further improve these new classes of nanostructured catalysts/photocatalysts should be addressed: i) In contrast to the native systems, where the active sites are well-structured, leading to high binding affinities and defined steric orientations of the substrates with respect to the catalytic/photocatalytic sites, the present aptamer systems are flexible and dynamic.[172][173][174] Adopting these tools to further en- gineer the artificial nucleic acid frameworks could substantially enhance their chemical functions.ii) At present, the examples of "nucleoapzymes", "aptananozymes" and "photoaptazymes" are quite limited.
Enhancing the diversity of systems by introducing additional chemical compositions and chemical transformations is important to develop the breadth of the field.In this context, the mosaic of artificial catalysts/photocatalysts lacks the important "Photoaptananozyme" brick (Figure 10).That is, supramolecular assemblies consisting of heterogenous photoactive-aptamer conjugates acting as photosynthetic model systems are still unrealized.The integration of aptamers with semiconductors quantum dots or the binding of aptamers [175] to photoactive metal-ion modified nanozymes, for example, Ru(II)-C 3 N 4 nanoparticles [176] could provide a means to achieve these goals.iii) Finally, the theoretical understanding of the structurefunction relationships in these nanosystems still remains vague.Although recent reports addressed computational modeling of spatial aptamer-ligand interactions, [177,178] the further development of theoretical tools to understand, and even predict, the activity of these systems, is mandatory.
Nonetheless, we are confident that the scientific community will rise to the above challenges, and eagerly await reports of new developments in this exciting field that will no doubt emerge in due course.

Figure 2 .
Figure 2. A) Schematic configurations of hemin/G-quadruplex-DBA "nanozymes" for the H 2 O 2 -promoted oxidation of dopamine to aminochrome.B) Rates of H 2 O 2 -promoted oxidation of dopamine to aminochrome in the presence of variable concentrations of dopamine using different nanozyme catalysts: a) hemin/G-quadruplex conjugated to DBA by a single "A" base, configuration I; b) hemin/G-quadruplex tethered to 5′-end DBA by a TATA spacer, configuration II; c) hemin/G-quadruplex tethered to 3′-end DBA by a TATA bridge, configuration III, d) configuration V consisting of scrambled 5′-end DBA conjugated to the hemin/G-quadruplex; e) separated hemin/G-quadruplex and DBA, configuration, IV.C) Molecular dynamics simulated structures of nucleoapzymes in configurations II and III, respectively.A-C) Adapted with permission.[68]Copyright 2016, American Chemical Society.

Figure 3 .
Figure 3. A) A set of Cu 2+ -terpyridine-modified DBA nucleoapzymes catalyzing the H 2 O 2 oxidation of dopamine to aminochrome.B) Rates of H 2 O 2 oxidation of dopamine in the presence of variable concentrations of dopamine using the Cu 2+ -terpyridine-functionalized nanozymes in configurations: a) III, b) I, c) IV, d) II, e) the Cu 2+ -terpyridine complex linked to a scrambled DBA, f) separated Cu 2+ -terpyridine complex and DBA.C) A set of Fe 3+terpyridine DBA catalyzing the oxidation of dopamine to aminochrome, in the presence of H 2 O 2 .A-C) Reproduced with permission.[69]Copyright 2018, American Chemical Society.D) A set of bis-Zn 2+ -pyridiyl-salen-type complexes conjugated to ATP aptamers, acting as nucleoapzyme, catalyzing the hydrolysis of ATP to ADP. e) Rates of hydrolysis of ATP in the presence of different concentrations of ATP using the bis-Zn 2+ -pyridiyl-salen-type complex in the configuration: a) III, b) I, c) IV, d) II, e) V. D-F) Reproduced with permission.[70]Copyright 2020, Wiley-VCH.

Figure 4 .
Figure 4. A) An imidazole-modified cholic acid aptamer conjugate acting as a nucleoapzyme for the catalyzed hydrolysis of a coumarin-functionalized choline ester, XII.B) Time-dependent fluorescence changes upon hydrolysis of the coumarin-functionalized choline ester, XII, in the presence of the imidazole-modified choline-aptamer nucleoapzyme conjugate (5 μm) (a) and 5 μM imidazole without aptamer (b).A,B) Reproduced with permission.[79]Copyright 2018, Royal Society of Chemistry.C) Schematic stepwise oxygen insertion into the Ar-H bond of tyrosinamide and the subsequent oxidation of the catechol product into amidodopachrome, in the presence of a set of nucleoapzymes consisting of Fe 3+ -terpyridine-functionalized tyrosinamideaptamer nucleoapzyme conjugated XX-XXIII, in the presence of H 2 O 2 and ascorbic acid.D) Rates of tyrosinamide oxidation in the presence of variable concentrations of the tyrosinamide substrate using the nucleoapzymes in configurations XX-XXIII and control systems corresponding to the Fe 3+catalyst conjugated to the scrambled bare sequence of the tyrosinamide-aptamer, configuration V, and the separated Fe 3+ -terpyridine catalyst and the tyrosinamide-aptamer. e) ESR spectrum corresponding to the ascorbate radical and hydroxyl radical generated by treatment of the H 2 O 2 and ascorbic acid mixture with the Fe 3+ -terpyridine/tyrosinamide-aptamer conjugate, and suggested mechanism for the oxygen-insertion into the Ar-H bond.C-E) Reproduced with permission.[80]Copyright 2019, Wiley-VCH.

Figure 5 .
Figure 5. A) Synthesis of DBA-functionalized Cu 2+ -ion-modified C-dots acting as aptananozymes for the catalyzed oxidation of dopamine by H 2 O 2 to yield aminochrome.Structures I-V represent schematically different aptananozyme configurations.B) Rates of dopamine oxidation by H 2 O 2 , 5 mm, in the presence of variable dopamine concentrations, using the different aptananozyme configurations and control systems.C) Application of the TBAfunctionalized Cu 2+ -modified C-dots as aptananozymes for the oxygen insertion into the Ar-H bond of tyrosinamide substrate, and the subsequent oxidation of the intermediate catechol product to amidodopachrome using H 2 O 2 /ascorbate as oxidation mixture in the presence of the aptananozyme.The structures X-XIV represent schematic configurations of the different aptananozymes.D) Rates of tyrosinamide oxidation by H 2 O 2 , 5 mm, and ascorbate, 5 mm, in the presence of variable tyrosinamide concentrations, using the different aptananozyme configurations and control systems.c) ESR spectra of the intermediate ascorbate radical and •OOH generated by the Cu 2+ -ion-modified C-dots aptananozymes X in the presence of the ascorbic acid/H 2 O 2 mixture.F) Schematic mechanistic cycle for the generation of the intermediate radical species by the aptananozymes.A-F) Adapted with permission.[135]Copyright 2021, American Chemical Society.

Figure 6 .
Figure 6.A) Synthesis of the polyadenine (pA)/DBA-functionalized AuNPs acting as aptananozymes for Panel I, the catalyzed oxidation of dopamine to aminochrome by H 2 O 2 and Panel II, the glucose-driven cascaded oxidation of dopamine to aminochrome via the primary catalyzed aerobic oxidation of glucose to gluconic acid and H 2 O 2 .B) Schematic configurations of the pA/dopamine-aptamer-modified AuNPs aptananozyme structures for the oxidation of dopamine by H 2 O 2 or aerobic glucose cascaded oxidation of dopamine, according to Panel I and Panel II, respectively.C) Rates of dopamine oxidation by H 2 O 2 , 5 mm, in the presence of variable dopamine concentrations, using the aptananozymes I-V and the respective control systems.D)Rates of dopamine oxidation to aminochrome in the presence of variable dopamine concentrations using the aerobic oxidation of glucose, 50 mm, by the bioreactor aptananozyme systems I-V and respective control systems.A-E) Adapted with permission.[136]Copyright 2022, American Chemical Society.
Figure 7C, panels I and II).Upon treatment of the MDA-MB-231 cells with a dose of 1.5 nm of AS1411-aptamer pA-AuNPs for a time-interval of two days ≈75% of cancer cell death was observed, whilst the normal MCF-10A epithelial breast cells were unaffected under these conditions (Figure 7C, panel I).Similarly, in vivo experiments using MDA-MB-231-breast-cancer-bearing mice demonstrated the effective chemodynamic cytotoxicity of the AS1411-aptamer-pA-AuNPs toward the cancer tumors (Figure 7C, panel II).

Figure 7 .
Figure 7. A) Schematic permeation of the polyadenine-stabilized AS1411-aptamer-conjugated AuNPs into the nucleolin-receptor-functionalized MDA-MB-231 breast cancer cells where intracellular catalyzed glucose-mediated generation of ROS agents and chemodynamic treatment of the cancer cells proceeds.B) Time-dependent formation of ROS agents visualized through the ROS-mediated fluorescence of di(acetoxymethyl ester)-6-carboxy-2′,7′dichlorodihydrofluorescein diacetate in non-cancerous epithelial breast cells (a) and breast cancer cells (b).C) Panel I: Cytotoxicity of different doses of non-aptamer modified pA-AuNPs treated MCF-10A epithelial cells (blue) and MDA-MB-231 breast cancer cells (red), b1 (0.9 nm), b2 (1.2 nm), and b3 (1.5 nm), respectively, for a time interval of two days, and of different doses of AS1411-aptamer-modified pA-AuNPs treated MCF-10A epithelial cell (blue) and MDA-MB-231 breast cancer cells (red), c1 (0.9 nm), c2 (1.2 nm), and c3 (1.5 nm), for a time interval of two days.Columns (a) corresponding to the control system: non-treated cells after two days.Panel II, In vivo time-dependent growth of xenograft-bearing MDA-MB-231 tumors treated with: a) the pA-AuNPs; b) pA-AuNPs conjugated to the scrambled bare AS1411-aptamer; c) pA-AuNPs conjugated to the AS1411-aptamer.(Mice are repeatedly treated at time intervals of 2-3 times per week.Error bars deduced from N = 3 mice samples).Right: Representative dimensions of tumors extracted from the respective mice samples.All results are presented as mean ± S.E.M. Significant results were evaluated using a t-test; *P < 0.05, **P < 0.01.A-C) Adapted with permission.[136]Copyright 2022, American Chemical Society.

Figure 8 .
Figure 8. A) Schematic configuration of a photosensitizer-aptamer/electron-acceptor supramolecular photosystem ("photoaptozyme") stimulating vectorial photoinduced electron transfer and FNR-mediated synthesis of NADPH or Pt NPs catalyzed H 2 -evolution.B) Top: Schematic configurations of four photosensitizer-tyrosinamide-aptamer conjugates, I-IV, where the photosensitizer is composed of Ru(II) tris-bipyridine and bipyridinium-tethered tyrosinamide, TA-MV 2+ , acts as the electron-acceptor ligand that binds to the aptamer constituent.Bottom: Photoaptazymes-driven photosensitized electron-transfer process leading to the catalyzed generation of NADPH or H 2 , in the presence of Na 2 EDTA as a sacrificial electron donor.C) Absorption spectra of NADPH generated by the different photoaptazymes at time intervals of irradiation of the photosystem outlined in (B), Na 2 EDTA 2 mm.D) The time-dependent concentration of NADPH generated by the photoaptazymes (or control systems) using the photosystems displayed in (B): a) IV III c) II d) I. e) The photosensitizer linked to the scrambled of the tyrosinamide-aptamer. f) The separated photosensitizer and tyrosinamide constituents.For all experiments the concentrations of photoaptazyme: 1 μm, TA-MV 2+ 20 μm, and Na 2 EDTA 20 mm.A-E) Adapted with permission.[174]Copyright 2019, Wiley-VCH.

Figure 9 .
Figure 9. TA-aptamer constituent (1 μm) 175 .Schematic configuration of the Zn(II) protoporphyrin IX (Zn(II)PPIX)/G-quadruplex photosensitizer conjugated to the TA-MV 2+ aptamer as a functional photoaptazyme acting as a photosystem for the vectorial photoinduced electron transfer to TA-MV 2+ and the subsequent FNR-catalyzed synthesis of NADPH.Photoaptazyme configurations include structures I-IV.B) Photosensitized electron transfer driven by the Zn(II)PPIX/G-quadruplex tyrosinamide-aptamer/TA-MV 2+ photosystem to yield TA-MV + • under illumination, in the presence of 2-mercaptoethonol as sacrificial electron donor.C) Absorption spectra of TA-MV + • generated upon operation of the photosystems shown in (A) by photoaptazymes: Panel I: configuration III; Panel II:: configuration IV; Panel III: configuration I; and Panel IV: configuration II.D) Time-dependent absorbance of TA-MV + • generated by the photosystems shown in (A) using different photoaptazymes (or control systems): a) photoaptazyme III, b) photoaptazyme IV, Figure 9H, curve (a) depicts the rate of photosensitized NADPH formation by the photoaptazymes Zn(II)PPIX/G-quadruplex-TA aptamer chains in comparison to the Zn(II)PPIX/G-quadruplex-bi-aptamer subunit, curve (b), and in comparison to the separated Zn(II)PPIX/G-quadruplex and TA-constituents, curve (c).In all experiments the concentration of the Zn(II)PPIX/G-quadruplex and TA components were identical.