The conjugation of catalytic sites to sequence-specific, ligand-binding nucleic acid aptamers yields functional catalytic ensembles mimicking the catalytic/binding properties of native enzymes. These catalyst-aptamer conjugates termed ‘nucleoapzymes’ reveal structural diversity, and thus, vary in their catalytic activity, due to the different modes of conjugation of the catalytic units to the nucleic acid aptamer scaffold. The concept of nucleoapzymes is introduced with the assembly of a set of catalysts consisting of the hemin/G-quadruplex DNAzyme (hGQ) conjugated to the dopamine aptamer. The nucleoapzymes catalyze the oxidation of dopamine by H2O2 to yield aminochrome. The catalytic processes are controlled by the structures of the nucleoapzymes, and chiroselective oxidation of l-DOPA and d-DOPA by the nucleoapzymes is demonstrated. In addition, the conjugation of a Fe(III)-terpyridine complex to the dopamine aptamer and of a bis-Zn(II)-pyridyl-salen-type complex to the ATP-aptamer yields hybrid nucleoapzymes (conjugates where the catalytic site is not a biomolecule) that catalyze the oxidation of dopamine to aminochrome by H2O2 and the hydrolysis of ATP to ADP, respectively. Variable, structure-controlled catalytic activities of the different nucleoapzymes are demonstrated. Molecular dynamic simulations are applied to rationalize the structure-catalytic function relationships of the different nucleoapzymes. The challenges and perspectives of the research field are discussed.
Enzymes, the natural biological catalysts, reveal unique catalytic efficiency mainly due to their ability to provide a specific binding site concentrating the substrate in close proximity to a reactive center catalyzing its transformation. Substrate selectivity and specificity and the ability to direct the substrate towards the catalytic center are achieved by the complex tertiary structure and the folding of the proteins. The isolated catalytic sites of enzymes and most of the existing bioinspired catalysts exhibit, however, significantly lower catalytic activities as compared with native enzymes. Inspired by nature, we developed a versatile concept to apply nucleic acid-based structures to mimic natural enzymes.
DNA aptamers are relatively stable, short and sequence-specific nucleic acids that bind to proteins or low-molecular-weight biomolecules (e.g. thrombin, dopamine, VEGF, ATP) [1–3]. Many aptamers fold into defined structures, e.g. G-quadruplexes that bind to specific domains of the protein ligand , or build distorted hairpin structures around a ligand [5,6] creating a selective binding site stabilized by hydrogen bonds , electrostatic interactions , and/or π–π-interactions (Figure 1A) . Although the structures of DNA aptamers are way beyond the complex structures of proteins, they reveal high binding affinities to their ligands and provide chiroselective binding sites. Indeed, the recognition properties of aptamers were applied for selective sensing [10–12] and a wide range of nanotechnological applications [13–16]. Another class of short nucleic acid sequences, widely used in biotechnology, are DNAzymes [17,18]. DNAzymes are usually sequence-specific nucleic acids that rearrange in the presence of metal ions, metal porphyrins, or organic ligands acting as cofactors, into catalytically active nanostructures [19–22]. One of the most studied DNAzymes consists of a guanosine-rich sequence that reconfigures in the presence of K+-ions, into a K+-ion stabilized G-quadruplex that binds the Fe(III)-hemin cofactor. This hemin/G-quadruplex (hGQ) nanostructure acts as a catalyst mimicking the function of native horseradish peroxidase [23,24]. Beyond the selection of sequence-specific nucleic acids acting as DNAzymes, nucleic acids acted as scaffold for the immobilization of metal ions or organic cofactors operating as supramolecular structures for catalysis and chiroselective catalysis [25–28]. Having in hand substrate-binding nucleic acid sequences (aptamers) and catalytic nucleic acid sequences (DNAzymes) or catalytic transition metal complexes, broadly applied in homogeneous catalysis, one can design functional conjugates (nucleoapzymes) that bind and concentrate the substrate close to a catalytic center, and thus increase the catalytic rate of the integrated catalyst. That is, the concentration of the substrate by the aptamer unit in proximity to the catalytic site increases the effective molarity at the active center, similar to this phenomenon in native enzymes. A diversity of possibilities to conjugate the catalyst to the aptamer exists: The aptamer forms a three-dimensional structure around the substrate such that the catalyst can be linked to different positions of the aptamer sequence to reach closest proximity to the substrate (5′-end, 3′-end, middle of the sequence, Figure 1B I, II, III). The insertion of a nucleic acid spacer of variable lengths between the catalytic unit and the aptamer-binding site might further increase the flexibility and diversity of the conjugates and the substrate accessibility by the catalyst (Figure 1B IV, V) . Although the three-dimensional structures of aptamer-ligand complexes are usually unknown, the identification of the aptamer nucleic acid bases interacting with the guest ligand can often be elucidated by NMR-spectroscopy. This fundamental information might then be applied to visualize, by appropriate molecular dynamic simulations (e.g. by the YASARA software), three-dimensional nanostructures of the nucleoapzymes [30,31]. By the evaluation of the dynamically simulated catalyst-aptamer conjugated constructs, the structure-catalytic function dependency of the nucleoapzymes is rationalized. It should be noted that the nucleoapzymes represent programmable catalytic nanostructures mimicking native enzymes. Their incorporation as ingredients of dynamic networks  and their integration into nanostructures, such as polymersomes, vesicles or microcapsules, could yield synthetic cell analogs.
Schematic configuration of catalyst-aptamer conjugates-nucleoapzymes.
The concept of nucleoapzymes is addressed by introducing different conjugates consisting of the dopamine-binding aptamer and a hGQ DNAzyme catalyzing the oxidation of dopamine to aminochrome by H2O2 . We demonstrate that the catalytic activities of the different DNAzyme-conjugates can be explained by the three-dimensional structures of the conjugates, and discuss the advantages of exchanging DNAzymes with catalytic transition metal complexes to create hybrid nucleoapzymes.
The native horseradish peroxidase, HRP, catalyzes the H2O2-mediated oxidation of dopamine (1) to aminochrome (2). The hGQ DNAzyme mimics HRP and catalyzes also the oxidation of dopamine to aminochrome by H2O2 (Figure 2A). The oxidation capability of hGQ is proved, yet it is inefficient. To enhance the catalytic activity of the hGQ DNAzyme towards the oxidation of dopamine, the concept of nucleoapzymes was applied by conjugating the hGQ catalytic center to the dopamine aptamer as a means to concentrate the substrate in spatial proximity to the catalytic site.
The catalyzed oxidation of dopamine by the nucleoapzyme consisting of the hGQ-dopamine aptamer conjugate.
This principle will be explained on two representative nucleoapzyme-conjugates where hGQ was bound to the 5′-end and to the 3′-end of the aptamer through a TATA-spacer (thymidin (T), adenosin (A) spacer), respectively (Figure 2B I, II). All nucleoapzyme-conjugates tested revealed higher catalytic efficiencies than hGQ alone and showed the typical saturation kinetics of enzymes. The oxidation rates of dopamine by the conjugates were substantially higher compared with the separated hGQ and aptamer units, yet they are dependent on the position of hGQ on the aptamer. For example, while the DNAzyme bound with a TATA-linker to the 5′-end of the aptamer (I) showed a 20-fold increase in activity compared with the separated compounds, the DNAzyme bound with a TATA-linker to the 3'-end of the aptamer (II) showed only a 3-fold increase in activity (Figure 2C).
Molecular dynamic simulations using the YASARA structure software package were used to account for the different catalytic activities of the nucleoapzymes . Thus, we provide a computational means to probe the structure-catalytic function relationship of the nucleoapzymes. The molecular dynamic simulations of the different hGQ/dopamine aptamer conjugates identified the energetically favored structures of the nucleoapzymes, and these defined the distances and orientations of the catalytic hGQ unit in respect to the substrate (dopamine)-binding site. The distances separating the catalytic sites from the binding sites dictate the frequency and probability of intimate contacts between the catalytic site and the substrate bound to the aptamer. Beyond the spatial and dynamic features that control the catalytic functions, the accessibility of the catalytic site towards the binding pocket is also visualized by the molecular dynamic simulations. In the case of the dopamine aptamer, dopamine is captured in a barrel-shaped binding pocket with a wide rim facing the 5′-end and a narrow rim facing the 3′-end of the aptamer. The computational simulations reveal for nucleoapzyme I, where the catalytic hGQ site is linked to the 5′-end of the aptamer, that the distance separating the catalyst from the binding site is 3–5 nm, and that the catalyst faces the substrate from the wider rim of the binding pocket. In turn, for the less efficient nucleoapzyme II that consists of the hGQ catalytic site conjugated to the 3′-end, the distance separating the catalytic site from the aptamer-binding domain is 9–15 nm, and the catalyst is oriented toward the narrow rim of the binding pocket (Figure 2D). While the distances separating the catalytic site from the substrate-binding domain play an important role in controlling the activities of the nucleoapzymes, it was found that linking the catalytic site to the 5′-end of the aptamer through a TATA-spacer improved the activity of the nucleoapzyme, compared with a nucleoapzyme where the hGQ catalytic site was only linked to the aptamer through a single adenosine base (kcat(TATA-spacer) = (18.3 ± 0.9) × 10−3 s−1; kcat(adenosine spacer) = (13.0 ± 0.7) × 10−3 s−1). This was attributed to the enhanced flexibility of the catalytic chain that allowed an improved fit between hGQ and the dopamine bound to the aptamer.
Beyond the structure-catalytic function relationship of the nucleoapzymes, the intrinsic chiral properties of the aptamer enable a chiroselective oxidation of catechol derivatives by the catalytic hGQ. The diastereomeric relations of l-DOPA and d-DOPA toward the dopamine-binding site resulted in enhanced oxidation of l-DOPA as compared with d-DOPA, by H2O2 to form dopachrome (l-DOPA: kcat = (10.1 ± 0.4) × 10−3 s−1; d-DOPA: kcat = (5.2 ± 0.4) × 10−3 s−1). While the chiroselectivity demonstrated by the nucleoapzyme-catalyzed oxidation of l/d-DOPA is modest, further modification of the aptamer sequence could enhance the chiroselective features of the system. The concept of hGQ-conjugated aptamers acting as nucleoapzymes was further extended to develop three different hGQ/N-hydroxy-l-arginine-aptamer nucleoapzymes. For all hGQ/aptamer conjugates, enhanced oxidation of N-hydroxy-l-arginine to citrulline by H2O2 was demonstrated, compared with the separated hGQ and aptamer units.
A major disadvantage of the DNAzyme/aptamer conjugates as catalytic ensembles relates, however, to the difficulty to identify couples of available DNAzymes and sequence-specific aptamers that bind the respective substrates for a bigger variety of transformations. Homogeneous catalysis provides, however, a rich arsenal of transition metal complexes that catalyze numerous chemical transformations. Thus, the conjugation of homogeneous catalysts to sequence-specific aptamers provides a versatile means to tailor hybrid nucleoapzymes. This approach will be exemplified with the preparation of metal-ion-terpyridine (3) functionalized nucleoapzymes for the catalyzed oxidation of dopamine to aminochrome  (Figure 3A), and a bis-Zn2+-pyridyl-salen-type complex (4) conjugated to the ATP-aptamer as nucleoapzyme for the hydrolysis of ATP to ADP, (Figure 3D) . The Cu(II)-terpyridine or Fe(III)-terpyridine was conjugated to the 3′-end and the 5′-end of the dopamine aptamer directly or through a four-thymidine (4T) spacer, yielding a diversity of nucleoapzymes. Figure 3B exemplifies the superior catalytic activities of the Fe(III)-terpyridine/dopamine aptamer conjugate that includes the catalytic site linked through the 4T spacer to the 5′-end and the 3′-end of the dopamine aptamer (nucleoapzyme III and IV, respectively). The rates of oxidation of dopamine by the nucleoapzymes are 140-fold and 95-fold enhanced as compared to the separated Fe(III)-terpyridine complex and aptamer units. The differences in the activities of the two nucleoapzymes were rationalized by molecular dynamic simulations, Figure 3C, that indicated that the distances separating the Fe(III)-terpyridine catalytic site from the dopamine substrate binding site are 30 Å for nucleoapzyme III and 43 Å for nucleoapzyme IV.
Hybrid nucleoapzymes catalyzing dopamine oxidation and ATP hydrolysis.
In addition, the variability in chemical transformations mediated by nucleoapzyme systems was demonstrated with the synthesis of a set of bis-Zn2+-pyridyl-salen-type complexes conjugated directly or through a two-thymidine (2T) spacer to the ATP-aptamer. Figure 3E exemplifies the superior catalytic performance of the bis-Zn2+- pyridyl-salen-type complex linked through the 2T-spacer to the 5′-end of the ATP-aptamer (nucleoapzyme V) and the bis-Zn2+-pyridyl-salen-type complex linked through the 2T-spacer to the 3′-end of the ATP-aptamer (nucleoapzyme VI). While no hydrolysis of ATP by the separated bis-Zn2+-pyridyl-salen-type complex and the non-modified ATP-aptamer could be detected, the nucleoapzymes demonstrate effective catalytic performance, with a catalytic rate constant being 2.3-fold higher for nucleoapzyme VI, where the catalytic complex is linked to the 3′-end of the ATP-aptamer, compared with nucleoapzyme V, where the catalytic site is linked to the 5′-end of the aptamer-binding site (kcat(V) = 297 × 10−2 min−1; kcat(VI) = 688 × 10−2 min−1). Molecular dynamic simulations rationalized the differences in activities of the nucleoapzymes V and VI in terms of the distances separating the catalytic site from the ATP-binding site. The distance separating the catalytic site from the ATP-binding domain in V corresponds to 44 Å, whereas the separating distance between these two components in VI is shortened to 18 Å, leading to the enhanced catalytic properties of nucleoapzyme VI (Figure 3F).
Although substantial advances in developing nucleoapzymes were demonstrated, major challenges are ahead of us: (i) At present the catalytic rates of nucleoapzymes are modest as compared with native enzymes. The further modification of the aptamer units, and the application of molecular dynamic simulations to identify superior structures could be a promising path to follow. For example, the design of cooperative binding  to the aptamer site could improve the effective molarity of the substrate. (ii) It is important to identify additional nucleoapzyme-catalyzed reactions, particularly chemical transformations of enhanced complexities. Indeed; recent studies reported on the development of nucleoapzymes catalyzing oxygen-insertion into C–H bonds . In this system, Cu(II)- or Fe(III)-terpyridine complexes conjugated to the tyrosinamide aptamer acted as nucleoapzymes for the H2O2 catalyzed oxidation of l-tyrosinamide into amidated l -DOPA, that was further oxidized into amidodopachrome. (iii) It is important to further develop sequence-specific aptamers for low-molecular mass substrates to enhance the diversity of nucleoapzyme-catalyzed transformations. Particularly, the design of aptamers of defined binding domains is important to further strengthen the application of computational tools to elucidate the structure-function relationships of nucleoapzymes. (iv) It is important to broaden the concept of nucleoapzymes to new directions. For example, the programmability of the base-sequence of nucleic acid scaffolds could be applied to enable nucleoapzyme-cascades. Alternatively, the conjugation of photocatalytic units to aptamers could pave the way to design supramolecular structures mimicking photosynthesis. Furthermore, the integration of nucleoapzymes into cell-like nanostructures such as polymersomes, vesicles or microcapsules could lead to synthetic cell systems. The recent advances in the development of nucleoapzymes are thus anticipated to attract interdisciplinary research efforts in the areas of catalysis, photocatalysis and material science, and contribute to new facets to systems chemistry and systems biology.
This report summarized a recent approach to construct catalyst-modified aptamers, nucleoapzymes, as catalytic conjugates that mimic native enzymes.
The binding of the reaction substrate to the aptamer leads to steric proximity between the substrate and the catalytic site, thus resembling the active site/binding site functionalities of enzymes.
The different conjugation modes of the catalyst to the aptamer, directly or through spacer units of variable lengths and compositions, establish for each chemical transformation the diversity of nucleoapzyme structures exhibiting the chemical variability.
The discussion has addressed the concept by the conjugation of the hGQ DNAzyme to the dopamine aptamer or N-hydroxy-l-arginine aptamers to catalyze the H2O2- mediated oxidation of dopamine to aminochrome and of N-hydroxy-l-arginine to citrulline.
The conjugation of Fe(III)-terpyridine to the dopamine aptamer and of a bis-Zn2+- pyridyl-salen-type complex to the ATP-aptamer yielded hybrid nucleoapzymes that catalyze the oxidation of dopamine to aminochrome or the hydrolysis of ATP to ADP.
The ability to model the reactivity of the nucleoapzymes by molecular dynamic simulations was shown and suggests that pre-design of nucleoapzymes of superior catalytic properties will be feasible.
Our research on nucleoapzymes is supported by the Volkswagen Foundation, Germany.
The Authors declare that there are no competing interests associated with the manuscript.