Mimicking Functions of Native Enzymes or Photosynthetic Reaction Centers by Nucleoapzymes and Photonucleoapzymes

The covalent linkage of catalytic units to aptamer sequence-specific nucleic acids exhibiting selective binding affinities for substrates leads to functional scaffolds mimicking native enzymes, nucleoapzymes. The binding of the substrates to the aptamer and their structural orientation with respect to the catalytic units duplicate the functions of the active center of enzymes. The possibility of linking the catalytic sites directly, or through spacer units, to the 5′-end, 3′-end, and middle positions of the aptamers allows the design of nucleoapzyme libraries, revealing structure–functions diversities, and these can be modeled by molecular dynamics simulations. Catalytic sites integrated into nucleoapzymes include DNAzymes, transition metal complexes, and organic ligands. Catalytic transformations driven by nucleoapzymes are exemplified by the oxidation of dopamine or l-arginine, hydroxylation of tyrosine to l-DOPA, hydrolysis of ATP, and cholic acid-modified esters. The covalent linkage of photosensitizers to the tyrosinamide aptamer leads to a photonucleoapzyme scaffold that binds the N-methyl-N′-(3-aminopropane)-4,4′-bipyridinium-functionalized tyrosinamide to the aptamer. By linking the photosensitizer directly, or through a spacer bridge to the 5′-end or 3′-end of the aptamer, we demonstrate a library of supramolecular photosensitizer/electron acceptor photonucleoapzymes mimicking the functions of photosystem I in the photosynthetic apparatus. The photonucleoapzymes catalyze the photoinduced generation of NADPH, in the presence of ferredoxin-NADP+-reductase (FNR), or the photoinduced H2 evolution catalyzed by Pt nanoparticles. The future prospects of nucleoapzymes and photonucleoapzymes are discussed.

T he information encoded in the base sequence of nucleic acids has been used, in the past two decades, to apply the biopolymer as a functional material to develop new catalysts. 1 The discovery of native nucleic acids (ribozymes) sparked tremendous efforts to evolve synthetically sequence-specific RNA-or DNA-based catalysts (ribozymes or DNAzymes). 2,3 Indeed, a wide variety of catalytic nucleic acids catalyzing the nicking or ligation of nucleic acid substrates, using cofactordependent sequence-specific oligonucleotides (cofactor as a metal ion or amino acid), were reported. 4,5 These catalytic transformations are limited, however, to substrates that form duplex recognition complexes with their substrates. In addition, the hemin cofactor conjugated to different configurations of the G-quadruplex acts as a versatile horseradish peroxidase-mimicking DNAzyme that catalyzes the H 2 O 2mediated oxidation of organic substrates that yields, similar to the native peroxidase, chromophores (dyes) or fluorescent products 6−9 and generates chemiluminescence, in the presence of luminol. 10 Also, hemin/G-quadruplex DNAzymes were broadly applied to mimic peroxidase reactions, such as the H 2 O 2 -catalyzed oxidation of phenols, 11 thiols, 12 NADH, 13 or aniline. 14 During these transformations, no specific binding of the substrates to the catalytically active intermediates occurred, leading to limited turnover rates as compared to those of the native enzymes. A different approach for using nucleic acids as functional materials to develop catalysts has involved the direct linkage of metal ions to the oligonucleotide scaffolds or the binding of metal ions to ligands covalently tethered to the DNA scaffolds. Different catalyzed transformations, such as carbon−carbon bond formation, 15 Diels−Alder, 16−18 or Michael addition reactions, 19 and the phosphorylation of hydroxyl groups, 20 were demonstrated by these oligonucleotide/metal complex hybrids. Although impressive chiroselective yields induced by the chiral DNA ligands were demonstrated, the systems revealed low turnover rates due to the lack of engineered binding sites.
Aptamers are sequence-specific nucleic acids that exhibit selective binding properties with respect to low-molecular weight substrates and macromolecules. 21,22 These binding features of aptamers were used to develop sensors, 23−26 switches, 27−29 and stimuli-responsive drug carriers, 30,31 and the aptamers were applied as therapeutic agents. 32 −34 In addition, it was demonstrated that mutation of the base sequences of aptamers and the modification of aptamers with redox groups 35 or photoisomerizable 36 units can enhance or switch the binding properties of aptamers. These unique features of aptamers guided the development of a new class of nucleic acid biocatalysts as schematically outlined in Figure 1A. The covalent tethering of a catalytic unit to the chiral aptamerbinding sequence yields a hybrid structure that mimics the functions of native enzymes. 37 The aptamer-binding site concentrates the substrate in spatial proximity to the catalytic unit, thereby mimicking the fundamental features of native enzymes, where the concentration of the substrate in the proximity of the active catalytic unit provides nature's "reactivity secret". The possibility of tethering the catalytic site at the 3′-or 5′-end of the aptamer or in intrachain positions and the feasibility of tethering the catalyst to the aptamer via flexible bridges or linking the catalyst between split aptamer subunits ( Figure 1B) provide a library of possible catalysts, termed by us "nucleoapzymes", that are expected to reveal variable catalytic functions. 37 An attempt to understand the structure−function relationships within this set of nucleoapzymes by means of molecular dynamics simulations could eventually allow, in the future, the in silico design of superior nucleoapzymes. Different catalysts were anchored to aptamers to construct the nucleoapzymes, and these included DNAzymes or transition metal complexes. In this Perspective, we summarize recent advances in the development of nucleoapzymes and broaden the concept to photonucleoapzymes, where photocatalytic−aptamer conjugates mimic the functions of photosynthesis.
■ DNAZYME−APTAMER CONJUGATES AS NUCLEOAPZYMES Realizing that peroxidases catalyze the oxidation of dopamine (1) to aminochrome (2), Golub et al. prepared a series of hemin/G-quadruplex-dopamine aptamer nucleoapzymes 37 (Figure 2A). Figure 2B presents the rates of oxidation of the dopamine substrate (1) by H 2 O 2 in the presence of the hemin/ G-quadruplex-dopamine aptamer nucleoapzymes, where the catalyst is conjugated to the 5′-end of the aptamer through a TATA spacer (structure I, curve a), to the 5′-end of the aptamer through a single A base (structure II, curve b), or to the 3′-end of the aptamer through a TATA spacer (structure III, curve c). For comparison, the H 2 O 2 -catalyzed oxidation of the dopamine substrate (1) by the separated hemin/Gquadruplex and aptamer units (configuration IV) is shown in curve d. The different nucleoapzymes examined in the study demonstrated superior catalytic activities compared to the oxidation of dopamine (1) to aminochrome (2) by the separated units. Nonetheless, the catalytic activities of the nucleoapzymes revealed structure-controlled activities. While the nucleoapzyme in configuration I revealed a 20-fold catalytic enhancement as compared to the separated components, the nucleoapzyme in configuration III showed an only 3-fold catalytic enhancement as compared to the separated components. Also, the flexibility of the tethering chain linking the catalyst to the aptamer scaffold affected the catalytic activity of the resulting nucleoapzyme. The different nucleoapzyme revealed a Michaelis−Menten type kinetics, exhibiting saturation rates upon full occupation of the aptamerbinding sites: V max values of 13.5 ± 0.5, 9.6 ± 0.2, and 2.6 ± 0.2 nM s −1 and k cat values of (18.3 ± 0.9), (13.0 ± 0.7), and (3.1 ± 0.1) × 10 −3 s −1 for configurations I−III, respectively. As the binding affinities of dopamine (1) for the different nucleoapzymes were similar, the structures of the intermediate substrate complexes have a dominant effect on the catalytic performance of the nucleoapzymes.
Molecular dynamics (MD) simulations were used to rationalize the experimental differences in the catalytic activities of the nucleoapzymes in terms of energetically favored structures of the hemin/G-quadruplex-dopamine aptamer conjugates that define the distances and orientations of the catalytic sites with respect to the aptamer-binding site. These two parameters dictate the frequency and probability of intimate contact between the catalyst and the substrate associated with the binding site. Besides the spatial and dynamic relation between the catalytic units and the substratebinding site, the accessibility of the catalytic site to the binding site can be similarly visualized by MD simulations. Panel I of Figure 2C depicts the MD-simulated structure of the most active hemin/G-quadruplex-dopamine aptamer nucleoapzyme composed of the catalytic site linked through the TATA spacer to the 5′-end of the aptamer (configuration I). The catalytic site is separated from the aptamer wide rim pocket by a distance corresponding to 3−5 nm, leading to a short reaction distance and accessibility of the catalyst to the dopamine substrate (1) associated with the aptamer-binding site. The MD-stimulated structure of the least effective nucleoapzyme, nucleoapzyme in configuration III, where the catalytic site is Biochemistry pubs.acs.org/biochemistry Perspective linked to the 3′-end of the aptamer through the TATA tether, is shown in panel II of Figure 2C. The distance separating the catalytic site from the binding site is substantially longer (9−15 nm), and the catalyst is oriented toward the narrow rim of the binding site, where the association of the dopamine substrate (1) is perturbed. All of these structural features of the nucleoapzyme in configuration II lead to the lower activity of the hybrid conjugate. This concept of hemin/G-quadruplexaptamer catalytic conjugates was further applied to design nucleoapzymes for the H 2 O 2 -mediated oxidation of L-arginine to citrulline using the L-arginine aptamer as the binding site. 37 APTAMERS AS NUCLEOAPZYMES The available number of DNAzymes that can be conjugated to aptamers limits the broadening of the concept of a nucleoapzyme. The substitution of DNAzymes by metal− ligand complexes, which exhibit catalytic functions, as catalytic units conjugated to aptamer-binding sequences, may provide a versatile path to yield metal−ligand complex-functionalized aptamers as enzyme-mimicking hybrid systems. This concept has been exemplified with the development of a series of Cu(II)−terpyridine-modified dopamine aptamers and a series of Fe(III)−terpyridine-modified dopamine aptamers as nucleoapzymes that catalyze the H 2 O 2 -mediated oxidation of dopamine (1) to aminochrome (2). 38 Figure 3A depicts the Fe(III)−terpyridine-modified dopamine aptamer catalyzed oxidation of dopamine (1) to aminochrome (2) using the nucleoapzyme consisting of the Fe(III)−terpyridine catalyst tethered to the 5′-end of the aptamer through a 4T bridge, configuration I, and the nucleoapzyme composed of the catalyst linked to the 3′-end of the aptamer through the 4T bridge, configuration II. Figure 3B shows that the rates of dopamine oxidation by the nucleoapzymes in configurations I and II are 180-and 35-fold faster, respectively, than the rates of oxidation of the substrate by the separated catalyst and aptamer units (curves a and b vs curve c). Kinetic analysis of the nucleoapzymes activity reveals that the values of k cat and K M are 267 × 10 −4 s −1 and 33 ± 12 μM and 200 × 10 −4 s −1 and 39 ± 18 μM for configurations I and II, respectively. As the binding affinity of the substrate for the two nucleoapzymes was similar, the enhanced catalytic activity of configuration I was attributed to the higher catalytic performance of the catalytic site in configuration I. Indeed, MD simulations revealed that the Fe(III)−terpyridine catalytic site is closer (30 Å) to the dopamine-binding site in configuration I, panel I of Figure 3C, as compared to the longer spatial separation of the catalytic site from the substrate-binding site in configuration II (43 Å), panel II of Figure 3C, suggesting the increased probability of the flexible catalytic unit reacting with the substrate in nucleoapzyme configuration I ( Figure 3C).  In addition, the Fe(III)−terpyridine complex linked to the tyrosinamide aptamer was found to act as a nucleoapzyme that catalyzes the oxidation of tyrosinamide (3) to amidodopachrome (5) by H 2 O 2 ( Figure 4A). 39 This oxygen-insertion process occurred only in the presence of an ascorbic acid/ H 2 O 2 mixture. A set of Fe(III)−terpyridine-functionalized tyrosinamide aptamers, in which the catalytic site was attached directly to the 5′-and 3′-ends of the aptamer (configurations I and II, respectively) or through 4T bridging tethers (configurations III and IV), were assembled. In the primary step, the oxidation of tyrosinamide (3) to the amidated L-DOPA (4) proceeded, and this was followed by the oxidation of 4 to amidodopachrome (5). Similarly, the cascaded oxidation of tyrosine (6) to L-DOPA (7) and dopachrome (8) was driven by the Fe(III)−terpyridine-functionalized tyrosinamide aptamer. Figure 4B depicts the oxidation of tyrosinamide (3) by nucleoapzyme configurations III and IV, in the presence of a H 2 O 2 /ascorbic acid mixture, in comparison to oxidation of 3 by the separated catalyst and aptamer components (curves a and b vs curve c). A 100-and 80-fold enhanced oxidation of 3 by the nucleoapzyme in configurations III and IV was observed (for configuration III, k cat = 11.8 ± 0 s −1 and K M = 205 μM; for configuration IV, k cat = 9.1 ± 0.2 s −1 and K M = 1.93 μM). Mechanistic insight into the catalytic oxidation of tyrosinamide to the catechol product by the H 2 O 2 /ascorbic acid mixture was obtained by electron spin resonance (ESR) measurements ( Figure 4C). While the formation of •OH and ascorbate radical by the nucleoapzyme in configuration III is inefficient, in the presence of the H 2 O 2 / ascorbic acid mixture effective formation of the two radicals was detected ( Figure 4D). The effective formation of the two radicals was attributed to a mutual synergistic cooperative formation of the two radicals, where the small amount of ascorbate radicals reacts with H 2 O 2 to yield peroxo ascorbate and •OH. The peroxo ascorbate dissociates to ascorbate radical and •OH, and the resulting •OH re-forms the ascorbate ( Figure 4D). This chain reaction provides a constant supply of the reactive oxygen species, •OH, and ascorbate radical. The two reactive species participate then in the formation of the tyrosine radical that recombines with •OH.
Besides oxidative nucleoapzymes, hydrolytic metal−organic complexes linked to aptamer-binding scaffolds were reported. The catalytic hydrolysis of ATP to ADP by a set of nucleoapzymes consisting of bis-Zn 2+ -pyridyl-salen type complexes linked directly or through 2T spacers to the 5′end or 3′-end of the ATP aptamer (configurations I−IV) is shown in Figure 5A. 40 Figure 5B depicts the catalytic performance of the 3′-2T-catalyst-ATP aptamer conjugate (configuration III) and the 5′-2T-catalyst-ATP aptamer conjugate (configuration IV) in comparison to the separated catalyst and aptamer components (curves a−c, respectively). While the separated components do not show catalytic hydrolysis of ATP, the 3′-2T-catalyst-modified aptamer conjugate shows a superior catalytic rate compared to that of the 5′-end catalyst-modified aptamer (for configuration III, k cat = 688 × 10 −2 min −1 and K M = 38 ± 7 μM; for configuration IV, k cat = 297 × 10 −2 min −1 and K M = 33 ± 6 μM). As the binding affinities of ATP for the different nucleoapzymes were similar (K d = 19 μM), it was concluded that the difference in the catalytic rates of the nucleoapzyme in configurations III Biochemistry pubs.acs.org/biochemistry Perspective and IV originates from the favored catalytic activity of the bis-Zn 2+ -pyridyl-salen complex in nucleoapzyme configuration III. MD simulations of the energetically stabilized structures of the nucleoapzyme in configurations III and IV 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, as compared to the spatial separation of the catalytic site from the reaction site in the nucleoapzyme in configuration IV ( Figure 5C). While the distance separating the catalytic site from the reaction site in the nucleoapzyme in configuration III is 18 Å, panel I Figure  5C, the distance separating the catalytic site from the reaction site in nucleoapzyme configuration IV corresponds to 44 Å, panel II of Figure 5C.

■ LIGAND-FUNCTIONALIZED NUCLEOAPZYMES
Specific amino acids in proteins such as histidine, lysine, or glutamate, in the proximity of the active site of enzymes, often participate in the catalytic process within the catalytic center by providing Lewis acids or bases, or nucleophilic functionalities for the activation of the active site-bound reaction substrates. In the past, these functionalities were tethered to artificial receptors such as crown ethers 41,42 or cyclodextrins 43 as part of efforts to mimic native enzymes by supramolecular structures.
Similarly, low-molecular weight ligands were tethered to aptamers as part of efforts to develop nucleoapzymes. For example, imidazole was tethered to the sequence-specific aptamer recognizing cholic acid. The imidazole-functionalized aptamer (Dcat1) acted as a nucleoapzyme catalyzing the hydrolysis of coumarin-modified cholic acid ester (9) to cholic acid (10) (Figure 6). The catalyzed hydrolysis of 9 was 100fold faster than the hydrolysis of 9 by the separated aptamer and imidazole units. Kinetic characterization of the nucleoapzyme revealed Michaelis−Menten kinetics (k cat = 0.8 ± 0.1 h −1 , and K M = 26 ± 6 μM). A set of related nucleoapzymes that included the imidazole ligand at other positions of the aptamer was prepared. All nucleoapzymes revealed enhanced activity with respect to the hydrolysis of 9, as compared to the separated components, yet DCat1 exhibited superior hydrolytic activity among the nucleoapzymes, demonstrating structure−function relationships of the nucleoapzyme library. 44 ■ PHOTONUCLEOAPZYMES AS PHOTOSYNTHETIC

MODEL SYSTEMS
Mimicking photosynthesis by artificial means is one of the "holey-grail" challenges in science. 45,46 These efforts include attempts to mimic the primary electron transfer events of the native photosystems of the photosynthetic apparatus (Z-  The successful introduction of the nucleoapzyme concept that organizes the catalytic units and reaction substrate by means of an aptamer scaffold was adapted to organize supramolecular photonucleoapzyme structures, where the photosensitizer unit and electron acceptor unit are forced into spatial proximity by means of an aptamer scaffold. That is, the noncovalent supramolecular association of an acceptor with a photosensitizer-functionalized aptamer yields the primary intimate structure between the light-harnessing component and the primary electron acceptor unit, where effective photoinduced ET proceeds, similar to the primary events in the photosynthetic reaction center. Figure 7A introduces the concept of a photonucleoapzyme by the functionalization of the tyrosinamide aptamer with a Ru-(bppy) 3 2+ photosensitizer. 63 The association of the tyrosinamide ligand modified with the N-methyl-N′-(3-aminopropane)-4,4′-bipyridinium electron acceptor (11) with the aptamer scaffold generated the supramolecular photonucleoapzyme assembly in which effective intracomplex electron transfer occurs. The photogenerated redox products were subsequently coupled to photosynthetic transformations involving the synthesis of NADPH and the evolution of hydrogen. Four different photonucleoapzymes were prepared, where the Ru(bppy) 3 2+ photosensitizer was covalently linked directly to the 5′-and 3′-ends of the aptamer (configurations I and II, respectively) or bridged to the 5′-and 3′-ends through 4T spacers (configurations III and IV, respectively). Effective, nonlinear, intramolecular ET quenching within all photonucleoapzyme structures was demonstrated ( Figure 7B, curves a−d). The quenching efficiencies revealed structure depend- Biochemistry pubs.acs.org/biochemistry Perspective ence and decreased in the following order: IV > III > II > I. Nonetheless, the quenching efficiencies for all photosensitizeraptamer conjugates were substantially higher than those of the diffusional ET quenching process of the separated Ru(bppy) 3 2+ photosensitizer and the tyrosinamide aptamer in the presence of the tyrosinamide/bipyridinium quencher ( Figure 7B, curve e). The structure-controlled electron transfer quenching efficiencies within the set of photonucleoapzymes dictated the yields of the resulting tyrosinamide/bipyridinium radical cation, TA-MV +• , formed under steady-state irradiation in the presence of Na 2 EDTA as a sacrificial electron donor. The yields of TA-MV +· guided the yields of the subsequent catalyzed chemical transformations ( Figure 8A). For example, Figure 8B depicts the time-dependent TA-MV +• driven reduction of NADP + to NADPH catalyzed by ferredoxin-NADP + -reductase (FNR). The quantum yields for the formation of NADPH by the different photonucleoapzymes followed the primary ET quenching efficiencies: IV (4.1%) > III (3.6%) > II (2.9%) > I (2.6%). These were substantially higher than the quantum yield for the generation of NADPH by the separated Ru(bppy) 3 2+ and aptamer in the presence of TA-MV 2+ (<0.2%) (cf. Figure 8B, curve e). Also, the photosensitized generation of TA-MV +• was coupled to the Pt nanoparticle-catalyzed evolution of the hydrogen fuel ( Figure 8A). The time-dependent yields of H 2 produced by the different photonucleoapzymes are presented in Figure 8C. The H 2 evolution yields by all photonucleoapzyme nanostructures (configurations I−IV) are substantially higher than the H 2 evolution yield of the separated Ru(bppy) 3 2+ and aptamer system in the presence of TA-MV 2+ (curves a−d vs curve e in Figure 8C). The quantum yields of H 2 evolution followed the primary ET quenching efficiencies in the supramolecular photonucleoapzyme structures [IV (3.0%) > III (3.2%) > II (2.2%) > I (1.8%) versus the quantum yield of H 2 evolution by the separated components (0.31%)]. The concept of photonucleoapzymes was further expanded by designing a library of Zn(II)-protoporphyrin IX/G-quadruplex chromophore-tyrosinamide aptamer conjugates operating as photonucleoapzymes,

■ CONCLUSIONS AND PERSPECTIVES
The structural modification of aptamers with catalytic and photocatalytic units to yield nucleoapzymes and photonucleoapzymes proved to be a viable and versatile approach for mimicking native enzymes and photosynthetic reaction centers. Although substantial advances were demonstrated, important challenges are ahead of us. (i) At present, the catalytic performance of nucleoapzymes is substantially lower than that of native enzymes. For example, the k cat value of the ATPase-mimicking nucleoapzyme, configuration III, is ∼103fold lower than that of the native F 1 -ATPase. 65 Nonetheless, the advantages of the nucleoapzyme as compared to native enzymes should be mentioned. These include the stability of the nucleoapzymes as compared to the native enzyme and, specifically, the diversity and programmability of the nucleoapzyme with respect to diverse substrates dictated by the elicited aptamers. This programmability is nonexistent in native enzymes. Although substantial advances in the engineering of nucleoapzymes were demonstrated, several pathways to further improve the catalytic performance of nucleoapzymes may be suggested. The catalytic rates of the nucleoapzymes should be improved. This could be accomplished by enhancing the binding affinity of the substrates for the aptamer-binding site (increase in the local molarity). This may be achieved by the mutation of the aptamer-binding sites 66 and by chemical modules that allosterically stabilize substrate-aptamer complexes. 35 In addition, the programmed positioning of the catalytic sites with respect to the substrate-binding site by means of the DNA scaffold could be a general means of controlling the distance between the catalytic unit and the substrate-binding site. Indeed, such a programmable hemin/Gquadruplex/dopamine aptamer supramolecular "ruler" demonstrated distance-controlled catalytic functions. 67

Notes
The authors declare no competing financial interest.