Photosensitizer

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Artificial Photosynthesis with Electron Acceptor/Photosensitizer-Aptamer Conjugates Guofeng Luo, Yonatan Biniuri, Wei-Hai Chen, Ehud Neumann, Michael Fadeev, Henri-Baptiste Marjault, Anjan Bedi, Ori Gidron, Rachel Nechushtai, David Stone, Thomas Happe, and Itamar Willner Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b02880 • Publication Date (Web): 13 Aug 2019 Downloaded from pubs.acs.org on August 13, 2019

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Artificial Photosynthesis with Electron Acceptor/Photosensitizer-Aptamer Conjugates Guo-Feng Luo,1 Yonatan Biniuri,1 Wei-Hai Chen,1 Ehud Neumann,2 Michael Fadeev,1 HenriBaptiste Marjault,2 Anjan Bedi,1 Ori Gidron,1 Rachel Nechushtai,2 David Stone,1 Thomas Happe,3 and Itamar Willner1* 1

Institute of Chemistry and Center for Nanoscience and Nanotechnology, The Hebrew

University of Jerusalem, Jerusalem, 91904, Israel 2

Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

3

Fakultät für Biologie und Biotechnologie, AG Photobiotechnologie, Ruhr Universität Bochum,

Universitätsstraße 150, 44801 Bochum, Germany

ABSTRACT: Sequence-specific aptamers act as functional scaffolds for the assembly of photosynthetic model systems. The Ru(II)-tris-bipyridine photosensitizer is conjugated by different binding modes to the anti-tyrosinamide aptamer to yield a set of photosensitizeraptamer binding scaffolds. The N-methyl-N'-(3-aminopropane)-4,4'-bipyridinium electron acceptor, MV2+, is covalently linked to tyrosinamide, TA, to yield the conjugate TA-MV2+. The tyrosinamide unit in TA-MV2+ acts as a ligand for anchoring TA-MV2+ to the Ru(II)-tris-

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bipyridine-aptamer scaffold, generating the diversity of photosensitizer-aptamer/electron acceptor supramolecular conjugates. Effective electron transfer (ET) quenching in the photosynthetic model systems is demonstrated, and the quenching efficiencies are controlled by the structural features of the conjugates. The redox species generated by the photosensitizeraptamer/electron acceptor supramolecular systems mediate the ferredoxin-NADP+ reductase, FNR, catalyzed synthesis of NADPH, and the Pt-nanoparticle-catalyzed evolution of hydrogen (H2). The novelty of the study rests on the unprecedented use of aptamer scaffolds as functional units for organizing photosynthetic model systems.

KEYWORDS: Electron transfer; Biocatalysis; H2-evolution; NADPH; Nucleic acid.

Mimicking photosynthesis by artificial systems, as a means of solar energy conversion and storage, has been a scientific “holy-grail” for the past four decades.1-3 Numerous efforts were directed to develop molecular,4,5 supramolecular2,6-12 and nanomaterial-based systems13-17 that duplicate the vectorial photo-induced electron transfer and charge separation events occurring in the photosynthetic reaction centers. In addition, the utilization of the photo-generated redox species for the synthesis of fuels, e.g., H2 evolution18-20 or CO2 fixation,21,22 or for driving chemical transformations attracts major research efforts.23,24 Ingenious supramolecular assemblies and micro-heterogeneous systems have been introduced during the years to mimic the vectorial electron transfer proceeding in the photosynthetic reaction centers. These included the use of photosensitizer electron acceptor diads25,26 or triads,27,28 and donor-photosensitizeracceptor triads.29 Also, micellar,30,31 liposomes,32-34 or nanoparticle systems35,36 have been used as microhetergenous systems for controlling photo-induced electron transfer reactions. In

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addition, the photo-generated redox species mediated in the presence of nanoparticle catalysts, such as Pt,37-40 Pd,41 or Ru42 have been applied to mediate H2 evolution or CO2 fixation to formate or methane. Also, photosensitizer generated redox species were coupled to the catalyzed generation of the NADPH cofactors that mediated enzyme-catalyzed biotransformation analogous to the photosynthetic driven reactions.43-45 The information encoded in the base sequence of nucleic acids has been extensively used to assemble complex nucleic structures,46 to develop switchable reconfigurable assemblies,47-49 to construct DNA-based machines50-52 and to yield cascaded biocatalytic nanostructures.53-57 In addition, selection procedures for the preparation of sequence-specific nucleic acids recognizing molecular or macromolecular ligands (aptamers) were developed.58,59 These unique properties of aptamers were broadly applied to develop sensors,60-62 DNA machines, e.g., tweezers,63 reconfigurable DNA origami structures,64 and stimuli-responsive drug carriers.65-67 It is surprising, however, that the unique selective binding properties of aptamers were never used as functional scaffolds to organize supramolecular photosynthetic model systems. Recently, we introduced the concept of “nucleoapzymes” where catalyst/aptamer conjugates acted as scaffolds that mimic the functions of enzymes. The concentration of the aptamer ligand (substrate) in proximity to the catalytic site duplicated the enzyme active-site reflected by the concentration and optimal orientation of the substrate in vicinity of the catalytic site.68,69 This concept is now adapted to develop photosensitizer-aptamer conjugates as functional scaffolds to bind electron acceptor units. The resulting aptamer-aided photosensitizer-electron acceptor supramolecular complexes control the photosensitized electron-transfer processes in the resulting ensembles and stimulate the operation of photosynthetic model systems. Specifically, we describe the synthesis of a series of Ru(II)-tris-bipyridine (Ru(bpy)32+) functionalized-anti-tyrosinamide conjugates

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acting as scaffolds for the organization of the photosynthetic model systems. We modify tyrosinamide with the N-methyl-N'-(3-aminopropane)-4,4'-bipyridinium electron acceptor as the functional ligand that generates the supramolecular complexes with the Ru(II)-tris-bipyridinemodified aptamer conjugates. We discuss the photosensitized electron transfer (ET) in the supramolecular photosensitizer-aptamer/electron acceptor complexes and use the photogenerated redox species to drive photosynthetic transformations, e.g., the photo-induced synthesis of NADPH and the light-induced-catalyzed evolution of hydrogen (H2). It should be noted that the photosensitized ET process from excited Ru(II)-tris-bipyridine to bipyridinium electron acceptors, in the presence of a sacrificial electron donor and the utilization of the resulting bipyridinium radical cation as ET mediator for driving biocatalytic processes and stimulating H2-evolution has been addressed in numerous studies.70,71 The novelty of the present study rests, however, on unprecedented use of aptamers as functional scaffold to organize photosynthetic model systems, and on the use of the aptamer ligand (tyrosinamide) as functional anchor to tether the electron relay for assembling the diversity of supramolecular photosynthetic model systems. Figure 1(A) depicts the concept to construct the artificial photosynthetic models. A photosensitizer, e.g., Ru(bpy)32+, S, is covalently linked to the anti-tyrosinamide aptamer. An electron acceptor, A1, e.g., N-methyl-N'-(3-aminopropane)-4,4'-bipyridinium, is covalently tethered to the tyrosinamide ligand to yield TA-MV2+, (1). The latter product includes the ligand to anchor the electron acceptor in close proximity to the photosensitizer site. The resulting ternary complex generated between Ru(bpy)32+-tyrosinamide aptamer conjugate and TA-MV2+ is anticipated to yield effective static quenching of the excited photosensitizer and to yield the reduced bipyridinium radical cation (E˚ (Ru(bpy)33+/2+*) = -0.87 V and E˚ (TA-MV2+/+•) = -0.49

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V vs. NHE). The subsequent reduction of bulk solution-solubilized TA-MV2+ to TA-MV+• can, then, utilize the reduced relay for photosynthetic bioelectrocatalytic transformations. Note that the schematic photosensitizer-aptamer/TA-MV2+ structure represents a versatile model that may include a broad set of photosynthetic model structures. For example, we coupled the Ru(bpy)32+ photosensitizer to the 5'-end (structure I) or 3'-end (structure II) of the aptamer sequence. In addition, the photosensitizer was coupled to the 5'-end of the aptamer through a 4×thymidine (4×T) bridging units (structure III) or to the 3'-end of the aptamer through a 4×T bridging units (structure IV), Figure 1(B). In fact, the design of the photosensitizer-aptamer conjugates may be extended to many other supramolecular complexes, e.g., altering the tether length, incorporation of the photosensitizer within a split aptamer subunit configuration, conjugation of the aptamer to the photosensitizer via duplexes, and more. The electron transfer quenching of the Ru(bpy)32+ photosensitizer by bipyridinium electron acceptor was demonstrated in numerous studies. We thus examined the electron transfer quenching of the photosensitizer units in the conjugates I-IV. Figure 2, curves (a)-(d), show the Stern-Volmer plots corresponding to the quenching of the Ru(bpy)32+ photosensitizer by different concentrations of TA-MV2+ in the different photosensitizer conjugates. For comparison, curve (e) depicts the Stern-Volmer plot corresponding to the quenching of the Ru(bpy)32+ photosensitizer covalently linked to the 5'-end of a nucleic acid that includes a scrambled composition of the bases comprising the anti-tyrosinamide aptamer. Figure 2, curve (f) shows the Stern-Volmer plot corresponding to the quenching of the separated Ru(bpy)32+ photosensitizer in the presence of the anti-tyrosinamide aptamer and different concentrations of TA-MV2+. Several conclusions can be deduced from the quenching experiments: (i) The fluorescence quenching of the Ru(bpy)32+ photosensitizer in the photosensitizer-aptamer conjugates is significantly

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enhanced as compared to the diffusional quenching of the photosensitizer separated from the electron acceptor TA-MV2+, in the presence of the aptamer, curve (f). The quenching rateconstant of the separated Ru(bpy)32+ by TA-MV2+ corresponds to kq = 0.94 × 109 M-1 s-1. (ii) The quenching curves of all photosensitizer-aptamer conjugates by TA-MV2+ are non-linear, implying that a static effective quenching proceeds in the conjugated structures. (iii) The fluorescence quenching of the photosensitizer depends on the structure of the photosensitizeraptamer conjugates. Linkage of the photosensitizer to the 3'-end of the aptamer leads to enhanced quenching as compared to the conjugate where the photosensitizer is linked to the 5'end. The introduction of a spacer (4×T) between the photosensitizer and the aptamer 3'- or 5'-end yields conjugates of enhanced fluorescence quenching. (iv) The fact that the fluorescence quenching of the photosensitizer linked to scrambled base aptamer sequence is linear, inefficient, and very similar to the quenching of the separated components, kq = 1.5 × 109 M-1 s-1, suggests that the binding of TA-MV2+ to the aptamer units of conjugates I-IV leads to the effective nonlinear electron transfer quenching. The differences in the ET quenching effeciencies of the different Ru(bpy)32+-aptamer conjugates, I-IV, by TA-MV2+ (and the subsequent electron transfer cascades) follow the binding affinities of TA-MV2+ to the different conjugates (for a detailed discussion and experimental support, vide infra). The electron transfer quenching processes occurring in the different photosensitizer-aptamer systems were used for the photo-induced steady state accumulation of the reduced tyrosinamide bipyridinium radical cation TA-MV+•. Towards these goals, the different photosensitizer-aptamer systems were irradiated in the presence of TA-MV2+ and Na2EDTA as electron donor, pH = 7.4, Figure S1(A). In these systems, the electron transfer fluorescence quenching process leads to the redox products TA-MV+•, and the oxidation of the sacrificial electron donor (Na2EDTA) leads to

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the steady-state accumulation of TA-MV+•. Figure S1(B), panels I-VI show the spectra of TAMV+• generated in the different systems. The bands at λ = 394 nm and λ = 602 nm are the characteristic absorption bands of bipyridinium radical cations. Figure S1(C) shows the timedependent generation of TA-MV+• in the different systems. The yields of photogenerated TAMV+• follow the quenching efficiencies in the different photosensitizer-aptamer conjugates by TA-MV2+. As the concentration ratio of the Ru(bpy)32+-aptamer and the TA-MV2+ electron acceptor is 1:20, and since the concentration of TA-MV+• generated by the conjugate IV after an irradiation time-interval of 30 minutes is ca. 16 μM (ca. 80% of the total TA-MV2+), we conclude that the photosensitizer-aptamer conjugate recycled 16 times while generating TAMV+•. That is, the TA-MV+• is exchanged by solution solubilized TA-MV2+ that bind to the aptamer and release TA-MV+• to the solution. We then used the effective electron transfer quenching processes in the photosensitizer-aptamer conjugates by TA-MV2+ to drive biocatalytic and catalytic transformations. In the first system, the photogenerated TA-MV+• was coupled to the generation of NADPH, in the presence of ferredoxin-NADP+ reductase, FNR, Figure 3(A). Bipyridinium radical cation were reported to reduce NAD(P)+ to NAD(P)H in the presence of diaphorase or FNR, and thus, the photogenerated TA-MV+• could reduce NADP+ to NADPH. Figure 3(B), panel I to panel IV, and Figure S2 show the absorption spectra of NADPH generated upon irradiation at time intervals by the different photosensitizer-aptamer/TA-MV2+ systems. Figure 3(C) shows the time dependent concentrations of NADPH generated by the different systems. Evidently, the yields of NADPH follow the fluorescence quenching efficiencies of the photosensitizer by TA-MV2+. The quantum yields for the generation of NADPH by the different photosystems are summarized in Table 1. For example, Figure 3(C) shows that for photosystem IV after a time-interval of 18 min, 57 μM of NADPH was generated.

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Realizing that the concentration of the photosensitizer-aptamer conjugate and of the TA-MV2+ electron acceptor corresponded to 1 μM and 20 μM, we conclude that the photosensitizeraptamer conjugate and TA-MV2+ were recycled 57 and 2.9 times upon generating the NADPH, respectively. In the second system, we used the Ru(bpy)32+-aptamer conjugates and TA-MV2+ as photosystems for the light-induced evolution of hydrogen (H2). Numerous studies have applied bipyridinium radical cations as mediator stimulating the evolution of H2 in the presence of Pt nanoparticles (NPs) as catalyst. The redox potential of TA-MV2+ corresponds to -0.49 V vs. NHE and is adequate to evolve H2, Figure 4(A). Accordingly, the set of photosensitizer-aptamer conjugates and the TA-MV2+ electron acceptor, and the respective control experiments, were irradiated in the presence of Na2EDTA and the Pt NPs as H2-evolution catalyst. Figure 4(B) shows the rate of H2-evolution from the different photosystems. The most effective catalyst is the photosensitizer-aptamer conjugate IV. The quantum yield for H2-evolution using the conjugate IV as photocatalyst corresponds to 3.9%. The rates of H2-evolution by the different photosensitizer-aptamer conjugates are shown in Figure 4(B), and the quantum yields for the generation of H2 are summarized in Table 2. The quantum yields follow the order of electron transfer quenching of the photosensitizer units in the different conjugates. The yields of H2evolution by the separated Ru(bpy)32+ and the aptamer/TA-MV2+ units or by the photosensitizerscrambled-bases aptamer conjugate are very low. These results demonstrate the significance of binding of TA-MV2+ to the aptamer unit and emphasize the function of the photosensitizeraptamer/TA-MV2+ supramolecular complex on the H2-evolution process. Furthermore, control experiments indicated that all the components included in the systems are essential to drive the H2 evolution, and in the absence of Na2EDTA, the TA-MV2+ acceptor or the Pt NP catalysts, the

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H2-evolution process is prohibited. The quantum yield for H2 generation by the photocatalytic conjugates is comparable to the quantum yield reported for other Ru(bpy)32+ or dye sensitized processes in the presence of bipyridinium salts and Pt particles as catalysts.72,73 Nonetheless, as all systems include sacrificial electron donors the quantum yields are dominated by the concentration of the electron donor. In our systems, we used relatively low concentrations of the Na2EDTA sacrificial electron donor (20 mM) in order to identify the photocatalytic differences of the conjugates I-IV. Thus, we conclude that the conjugates III and IV lead to relatively high H2-evoluation quantum yields. The results have demonstrated that the photo-sensitized generation of NADPH or the H2evolution are enhanced in the presence of the Ru(bpy)32+-aptamer conjugates and TA-MV2+ as compared to the separated components. Furthermore, we observed differences between the different conjugates and the yields of the photo-sensitized generation of NADPH or H2 evolution follow the electron transfer fluorescence quenching of the photosensitizer within the different conjugates. The enhanced photo-induced biocatalytic and catalytic transformations within the conjugates were attributed to the binding of TA-MV2+ to the aptamer unit, a process that facilitated the photo-induced electron transfer within the supramolecular complexes. The different fluorescence quenching efficiencies demonstrated by the different conjugates might originate from different binding affinities to the conjugates and/or from different orientations of the TA-MV2+ electron acceptor in respect to photosensitizer unit. Recently, we have applied microscale thermophoresis (MST) as a versatile method to evaluate the association constant of ligands to their aptamers. The evaluation of the dissociation constants of aptamerligand complexes is based on the labeling of the complex with a fluorophore and probing the formation of the complex by following the fluorescence changes of the thermophoretic curves of

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the complexes as a result of the aptamer-ligand complexes. Realizing that the Ru(bpy)32+ photosensitizer in the different conjugates exhibits strong fluorescence, the thermophoretic curves generated by MST provide a direct means to evaluate the dissociation constants of the conjugates/TA-MV2+. Figure S3 and accompanying discussion shows the microscale thermophoretic curves observed upon the interaction of conjugate IV with different concentrations of TA-MV2+ and describes the evaluation of the dissociation constant of the conjugate IV/TA-MV2+. The derived Kd value of the conjugate IV/TA-MV2+ complex corresponds to 95 ± 17 nM. Similar MST experiments were performed to evaluate the dissociation constants of all supramolecular complexes generated between TA-MV2+ and the different Ru(bpy)32+-aptamer conjugates, and the values are summarized in Table 3. We find that, within the experimental errors, all Ru(bpy)32+-aptamer conjugates (I-IV) reveal identical Kd values, implying similar affinities for the association of the acceptor TA-MV2+. We thus conclude that presumably, the orientations of the electron acceptors relative to the photoactive centers are the origin for the different fluorescence quenching efficiencies. Indeed, fluorescence lifetime measurements, Figure 5, allow the quantitative assessment of the static quenching rate constants, ksq, associated with the supramolecular complexes I-IV. While the photosensitizeraptamer conjugates, in the absence of TA-MV2+, or the Ru(bpy)32+-scrambled aptamer conjugate in the presence of TA-MV2+, do not show any fluorescence quenching on short timescales, e.g. 0.05 μs, Figure S4, all photosensitizer-aptamer conjugates reveal rapid, first-orders, decay transient. From the fluorescence decay curves of conjugates I-IV shown in Figure 5, we evaluated the static fluorescence quenching rate constant, ksq, and these are detailed in Table 3. Clearly, the ksq for the conjugates follow the order IV > III > II > I, consistent with the order of

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activities of the photosensitizer-aptamer conjugates in the steady state biocatalytic and catalytic transformations. The results demonstrate that the introduction of the 4×T spacer units into the photosensitizeraptamer conjugates improve the quenching processes compared to the conjugates that include the Ru(bpy)32+ photosensitizer linked directly to the ends of the aptamer, e.g., III > I; IV > II. Presumably, the flexibility of the 4×T tether allows closer proximity between the photosensitizer and acceptor units, leading to the enhanced quenching. Although the structural features of tyrosinamide in the aptamer pocket are unknown and thus, molecular dynamic simulation identifying the spatial orientation of TA-MV2+ in respect to the photosensitizer units cannot be performed, our studies suggest the steric orientation of the quencher in respect to the photosensitizer units play a role in controlling the quenching processes in the supramolecular complexes of photosensitizer-aptamer/TA-MV2+. In conclusion, the study introduced sequence-specific aptamers as functional scaffolds for the assembly

of

photosynthetic

model

systems.

Specifically,

the

Ru(II)-tris-bipyridine

photosensitizer has been conjugated by different binding modes to the anti-tyrosinamide aptamer to yield a set of photosensitizer-aptamer binding scaffolds. The N-methyl-N'-(3-aminopropane)4,4'-bipyridinium electron acceptor, MV2+, was covalently-linked to the tyrosinamide, TA, to yield the electron acceptor/tyrosinamide conjugate, TA-MV2+, where the tyrosinamide ligand acted as an anchoring unit for linking TA-MV2+ electron acceptor to the Ru(II)-tris-bipyridineaptamer scaffolds to generate the diversity of photosensitizer/aptamer-bound electron acceptor supramolecular diads acting as photosynthetic model systems. Effective ET quenching within the supramolecular photosensitizer-aptamer/electron acceptor conjugate, as compared to the separated components, was demonstrated, and the ET quenching processes were controlled by

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the structure of the systems. The redox-species generated by the photosensitizer-aptamer/electron acceptors were coupled to the biocatalytic FNR synthesis of NADPH and to the Pt-nanoparticlecatalyzed evolution of hydrogen (H2). The most important result of the study is the demonstration that aptamers can be used as functional units to organize supramolecular photosynthetic model systems and that the ET processes within these assemblies are controlled by their structures. In addition, the availability of other aptamers, and the possibilities to program structures of nucleic acid, and to reconfigure their structures by external triggers, pave the way to construct photosystems of enhanced complexities (e.g., triads, tetrads) and to generate switchable photosynthetic model systems.

ASSOCIATED CONTENT Supporting Information. Details for materials preparation and additional experimental results. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Author Contributions G.-F. L. and Y. B. planed and performed the experiments. W.-H. C. and M. F. participated in the characterization of the conjugates. E. N., H.-B. M. and R. N. purified and characterized the FNR. A. B. and O. G. participated in the time-resolved fluorescence measurements. D. S. and W.-H. C. participated in the hydrogen evolution experiments. T. H. participated in the formulation of the photosensitizer/aptamer concept. I. W. formulated the photosensitizer/aptamer complex,

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participated in planning the experiments, mentored the research project and formulated the paper. All authors contributed to the final text of the paper. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT I.W. and T.H. gratefully acknowledge financial support from the Volkswagen Stiftung (Design of [FeS]-cluster containing Metallo-DNAzymes (Az 93412)). This work was further supported by

the

Cluster

of

Excellence

RESOLV

(EXC2033)

funded

by

the

Deutsche

Forschungsgemeinschaft (TH). We thank Prof. U. Banin, The Hebrew University, Jerusalem, for providing the H2 analysis instrument. REFERENCES (1) Gust, D.; Moore, T. A.; Moore, A. L. Solar fuels via artificial photosynthesis. Acc. Chem. Res. 2009, 42, 1890-1898. (2) Wasielewski, M. R. Photoinduced electron transfer in supramolecular systems for artificial photosynthesis. Chem. Rev. 1992, 92, 435-461. (3) Imahori, H. Porphyrin-fullerene linked systems as artificial photosynthetic mimics. Org. Biomol. Chem. 2004, 2, 1425-1433. (4) Li, C.; Wang, M.; Pan, J.; Zhang, P.; Zhang, R.; Sun, L. Photochemical hydrogen production catalyzed by polypyridyl ruthenium-cobaloxime heterobinuclear complexes with different bridges. J. Organomet. Chem. 2009, 694, 2814-2819. (5) Samuel, A. P. S.; Co, D. T.; Stern, C. L.; Wasielewski, M. R. Ultrafast photodriven intramolecular electron transfer from a zinc porphyrin to a readily reduced diiron hydrogenase model complex. J. Am. Chem. Soc. 2010, 132, 8813-8815.

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separation efficiency on the donor-acceptor interaction in photoinduced electron transfer Angew. Chem. Int. Ed. 2016, 55, 629-633. (14)Imahori, H.; Tamaki, K.; Guldi, D. M.; Luo, C.; Fujitsuka, M.; Ito, O.; Sakata, Y.; Fukuzumi, S. Modulating charge separation and charge recombination dynamics in porphyrin-fullerene linked dyads and triads:  marcus-normal versus inverted region. J. Am. Chem. Soc. 2001, 123, 2607-2617. (15)Yamamoto, M.; Föhlinger, J.; Petersson, J.; Hammarström, L.; Imahori, H. A ruthenium complex-porphyrin-fullerene-linked molecular pentad as an integrative photosynthetic model. Angew. Chem. Int. Ed. 2017, 56, 3329-3333. (16)Zhang, T.; Lin, W. Metal-organic frameworks for artificial photosynthesis and photocatalysis. Chem. Soc. Rev. 2014, 43, 5982-5993. (17)Degani, Y.; Willner, I. Photoinduced hydrogen evolution by a zwitterionic diquat electron acceptor. The functions of silicon dioxide colloid in controlling the electron-transfer process. J. Am. Chem. Soc. 1983, 105, 6228-6233. (18)Zhou, H.; Li, X.; Fan, T.; Osterloh, F. E.; Ding, J.; Sabio, E. M.; Zhang, D.; Guo, Q. Artificial inorganic leafs for efficient photochemical hydrogen production inspired by natural photosynthesis. Adv. Mater. 2010, 22, 951-956. (19)Tachibana, Y.; Vayssieres, L.; Durrant, J. R. Artificial photosynthesis for solar watersplitting. Nat. Photonics 2012, 6, 511-518. (20)Kitamoto, K.; Sakai, K. Pigment-acceptor-catalyst triads for photochemical hydrogen evolution. Angew. Chem. Int. Ed. 2014, 53, 4618-4622. (21)Willner, I.; Mandler, D.; Riklin, A. Photoinduced carbon dioxide fixation forming malic and isocitric acid. J. Chem. Soc.-Chem. Commun. 1986, 13, 1022-1024.

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(22)Yadav, R. K.; Baeg, J. O.; Oh, G. H.; Park, N. J.; Kong, K. J.; Kim, J.; Hwang, D. W.; Biswas, S. K. A photocatalyst-enzyme coupled artificial photosynthesis system for solar energy in production of formic acid from CO2. J. Am. Chem. Soc. 2012, 134, 11455-11461. (23)Kim, J. H.; Lee, M.; Lee, J. S.; Park, C. B. Self-assembled light-harvesting peptide nanotubes for mimicking natural photosynthesis. Angew. Chem. Int. Ed. 2012, 51, 517-520. (24)Mandler, D.; Willner, I. Photosensitized NAD(P)H regeneration systems; application in the reduction of butan-2-one, pyruvic, and acetoacetic acids and in the reductive amination of pyruvic and oxoglutaric acid to amino acid. J. Chem. Soc.-Perkin Trans. 2 1986, 6, 805-811. (25)Cormier, R. A.; Posey, M. R.; Bell, W. L.; Fonda, H. N.; Connolly, J. S. Synthesis and characterization of a directly linked porphyrin-anthraquinone molecule. Tetrahedron 1989, 45, 4831-4843. (26)Yonemoto, E. H.; Kim, Y.; Schmehl, R. H.; Wallin, J. O.; Shoulders, B. A.; Richardson, B. R.; Haw, J. F.; Mallouk, T. E. Photoinduced electron transfer reactions in zeolite-based donor-acceptor and donor-donor-acceptor diads and triads. J. Am. Chem. Soc. 1994, 116, 10557-10563. (27)Batova, E. E.; Levin, P. P.; Shafirovich, V. Y. Long-lived radical ion pair state of Znporphyrin-viologen-quinone triad. New J. Chem. 1990, 14, 269-271. (28)Borgström, M.; Shaikh, N.; Johansson, O.; Anderlund, M. F.; Styring, S.; Åkermark, B.; Magnuson, A.; Hammarström, L. Light induced manganese oxidation and long-lived charge separation in a Mn2II,II-RuII(bpy)3-acceptor triad. J. Am. Chem. Soc. 2005, 127, 17504-17515. (29)Collin, J. P.; Guillerez, S.; Sauvage, J. P.; Barigelletti, F.; De Cola, L.; Flamigni, L.; Balzani, V. Photoinduced process in dyads and triads: an osmium (II)-bis (terpyridine)

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photosensitizer covalently linked to electron donor and acceptor groups. Inor. Chem. 1992, 31, 4112-4117. (30)Amao, Y.; Maki, Y.; Fuchino, Y. Photoinduced hydrogen production with artificial photosynthesis system based on carotenoid-chlorophyll conjugated micelles. J. Phys. Chem. C 2009, 113, 16811-16815. (31)Turro, N. J.; Grätzel, M.; Braun, A. M. Photophysical and photochemical processes in micellar systems. Angew. Chem. Int. Ed. 1980, 19, 675-696. (32)Calvin, M. Artificial photosynthesis: Quantum capture and energy storage. Photochem. Photobiol. 1983, 37, 349-360. (33)Infelta, P. P.; Grätzel, M.; Fendler, J. H. Aspects of artificial photosynthesis. Photosensitized electron transfer and charge separation in cationic surfactant vesicles. J. Am. Chem. Soc. 1980, 102, 1479-1483. (34)Ford, W. E.; Otvos, J. W.; Calvin, M. Photosensitised electron transport across phospholipid vesicle walls. Nature 1978, 274, 507-510. (35)Willner, I.; Otvos, J. W.; Calvin, M. Photosensitized electron-transfer reactions in colloidal silicon dioxide systems: charge separation at a solid-aqueous interface. J. Am. Chem. Soc. 1981, 103, 3203-3205. (36)Willner, I.; Yang, J. M.; Laane, C.; Otvos, J. W.; Calvin, M. The function of silicon dioxide colloids in photoinduced redox reactions. Interfacial effects on the quenching, charge separation, and quantum yields. J. Phys. Chem. 1981, 85, 3277-3282. (37)Grätzel, M. Artificial photosynthesis: water cleavage into hydrogen and oxygen by visible light. Acc. Chem. Res. 1981, 14, 376-384.

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(38)Adar, E.; Degani, Y.; Goren, Z.; Willner, I. Photosensitized electron-transfer reactions in βcyclodextrin aqueous media: effects on dissociation of ground-state complexes, charge separation, and hydrogen evolution. J. Am. Chem. Soc. 1986, 108, 4696-4700. (39)Kiwi, J.; Grätzel, M. Projection, size factors, and reaction dynamics of colloidal redox catalysts mediating light induced hydrogen evolution from water. J. Am. Chem. Soc. 1979, 101, 7214-7217. (40)Wang, C.; deKrafft, K. E.; Lin, W. Pt nanoparticles@photoactive metal-organic frameworks: efficient hydrogen evolution via synergistic photoexcitation and electron injection. J. Am. Chem. Soc. 2012, 134, 7211-7214. (41)Willner, I.; Mandler, D. Characterization of palladium-β-cyclodextrin colloids as catalysts in the photosensitized reduction of bicarbonate to formate. J. Am. Chem. Soc. 1989, 111, 13301336. (42)Willner, I.; Maidan, R.; Mandler, D.; Dürr, H.; Dörr, G.; Zengerle, K. Photosensitized reduction of carbon dioxide to methane and hydrogen evolution in the presence of ruthenium and osmium colloids: strategies to design selectivity of products distribution. J. Am. Chem. Soc. 1987, 109, 6080-6086. (43)Mandler, D.; Willner, I. Solar light induced formation of chiral 2-butanol in an enzymecatalyzed chemical system J. Am. Chem. Soc. 1984, 106, 5352-5353. (44)Mandler, D.; Willner, I. Photoinduced enzyme-catalyzed synthesis of amino acids by visible light. J. Chem. Soc.-Chem. Commun. 1986, 11, 851-853. (45)Mandler, D.; Willner, I. Photochemical fixation of carbon dioxide: enzymic photosynthesis of malic, aspartic, isocitric, and formic acids in artificial media. J. Chem. Soc.-Perkin Trans. 2 1988, 6, 997-1003.

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(46)Wang, F.; Lu, C. H.; Willner, I. From cascaded catalytic nucleic acids to enzyme-DNA nanostructures: controlling reactivity, sensing, logic operations, and assembly of complex structures. Chem. Rev. 2014, 114, 2881-2941. (47)Wang, F.; Liu, X.; Willner, I. DNA switches: from principles to applications. Angew. Chem. Int. Ed. 2015, 54, 1098-1129. (48)Wu, N.; Willner, I. pH-stimulated reconfiguration and structural isomerization of origami dimer and trimer systems. Nano Lett. 2016, 16, 6650-6655. (49)Wang, J.; Yue, L.; Wang, S.; Willner, I. Triggered reversible reconfiguration of Gquadruplex-bridged “domino”-type origami dimers: application of the systems for programmed catalysis. ACS Nano 2018, 12, 12324-12336. (50)Goodman, R. P.; Heilemann, M.; Doose, S.; Erben, C. M.; Kapanidis, A. N.; Turberfield, A. J. Reconfigurable, braced, three-dimensional DNA nanostructures. Nat. Nanotechnol. 2008, 3, 93-96. (51)Teller, C.; Willner, I. Functional nucleic acid nanostructures and DNA machines. Curr. Opin. Biotechnol. 2010, 21, 376-391. (52)Seeman, N. C. From genes to machines: DNA nanomechanical devices. Trends Biochem. Sci. 2005, 30, 119-125. (53)Wilner, O. I.; Weizmann, Y.; Gill, R.; Lioubashevski, O.; Freeman, R.; Willner, I. Enzyme cascades activated on topologically programmed DNA scaffolds. Nat. Nanotechnol. 2009, 4, 249-254. (54)Fu, J.; Liu, M.; Liu, Y.; Woodbury, N. W.; Yan, H. Interenzyme substrate diffusion for an enzyme cascade organized on spatially addressable DNA nanostructures. J. Am. Chem. Soc. 2012, 134, 5516-5519.

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(55)Wang, Z. G.; Wilner, O. I.; Willner, I. Self-assembly of aptamer-circular DNA nanostructures for controlled biocatalysis. Nano Lett. 2009, 9, 4098-4102. (56)Liu, M.; Fu, J.; Hejesen, C.; Yang, Y.; Woodbury, N. W.; Gothelf, K.; Liu, Y.; Yan, H. A DNA tweezer-actuated enzyme nanoreactor. Nat. Commun. 2013, 4, 2127. (57)Xin, L.; Zhou, C.; Yang, Z.; Liu, D. Regulation of an enzyme cascade reaction by a DNA machine. Small 2013, 9, 3088-3091. (58)Deng, B.; Lin, Y.; Wang, C.; Li, F.; Wang, Z.; Zhang, H.; Le, X. C. Aptamer binding assays for proteins: the thrombin example-a review. Anal. Chim. Acta 2014, 837, 1-15. (59)Huizenga, D. E.; Szostak, J. W. A DNA aptamer that binds adenosine and ATP. Biochemistry 1995, 34, 656-665. (60)Willner, I.; Zayats, M. Electronic aptamer-based sensors. Angew. Chem. Int. Ed. 2007, 46, 6408-6418. (61)Famulok, M.; Mayer, G. Aptamer modules as sensors and detectors. Acc. Chem. Res. 2011, 44, 1349-1358. (62)Liu, X.; Wang, F.; Aizen, R.; Yehezkeli, O.; Willner, I. Graphene oxide/nucleic-acidstabilized silver nanoclusters: functional hybrid materials for optical aptamer sensing and multiplexed analysis of pathogenic DNAs. J. Am. Chem. Soc. 2013, 135, 11832-11839. (63)Elbaz, J.; Moshe, M.; Willner, I. Coherent activation of DNA tweezers: a “SET-RESET” logic system. Angew. Chem. Int. Ed. 2009, 48, 3834-3837. (64)Wu, N.; Willner, I. Programmed dissociation of dimer and trimer origami structures by aptamer-ligand complexes. Nanoscale 2017, 9, 1416-1422.

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(65)Chen, W. H.; Liao, W. C.; Sohn, Y. S.; Fadeev, M.; Cecconello, A.; Nechushtai, R.; Willner, I. Stimuli-responsive nucleic acid-based polyacrylamide hydrogel-coated metal-organic framework nanoparticles for controlled drug release. Adv. Funct. Mater. 2018, 28, 1705137. (66)Lu, C. H.; Willner, I. Stimuli-responsive DNA-functionalized nano-/microcontainers for switchable and controlled release. Angew. Chem. Int. Ed. 2015, 54, 12212-12235. (67)Liao, W. C.; Lilienthal, S.; Kahn, J. S.; Riutin, M.; Sohn, Y. S.; Nechushtai, R.; Willner, I. pH-and ligand-induced release of loads from DNA-acrylamide hydrogel microcapsules. Chem. Sci. 2017, 8, 3362-3373. (68)Golub, E.; Albada, H. B.; Liao, W. C.; Biniuri, Y.; Willner, I. Nucleoapzymes: hemin/Gquadruplex DNAzyme-aptamer binding site conjugates with superior enzyme-like catalytic functions. J. Am. Chem. Soc. 2016, 138, 164-172. (69)Biniuri, Y.; Albada, B.; Wolff, M.; Golub, E.; Gelman, D.; Willner, I. Cu2+ or Fe3+ terpyridine/aptamer conjugates: nucleoapzymes catalyzing the oxidation of dopamine to aminochrome. ACS Catal. 2018, 8, 1802-1809. (70)Harriman, A.; Mills, A. Optimisation of the rate of hydrogen production from the tris (2, 2′bipyridyl) ruthenium (II) photosensitised reduction of methyl viologen. J. Chem. Soc. Faraday Trans. 2 1981, 77, 2111-2124. (71)Kalyanasundaram, K. Photophysics, photochemistry and solar energy conversion with tris (bipyridyl) ruthenium (II) and its analogues. Coord. Chem. Rev. 1982, 46,159-244. (72)Prasad, D. R.; Hoffman, M. Z. Photodynamics of the tris (2,2'-bipyrazine) ruthenium (2+)/methylviologen/EDTA system in aqueous solution. J. Am. Chem. Soc. 1986, 108, 25682573.

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(73)Nenadović, M. T.; Mićić, O. I.; Rajh, T.; Savić, D. Temperature effect on the photoinduced reduction of methyl viologen with several sensitizers and the evolution of hydrogen from water. J. Photochem. 1983, 21, 35-44.

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Figure 1. (A) Scheme for the photo-sensitized electron transfer quenching within a photosensitizer-modified aptamer/electron acceptor supramolecular complex and the utilization of the photo-generated redox products for photosynthetic transformations, e.g. H2-evolution or the formation of the NADPH cofactor. The bipyridinium tethered tyrosinamide, TA-MV2+, (1), acts as electron acceptor that binds to the Ru(bpy)32+-aptamer conjugate. In this general scheme the A1 corresponds to the primary electron acceptor that quenches the photosensitizer by an electron acceptor. A2 represents a secondary acceptor that accepts the electrons from reduced A1 and mediates the electron transfer cascades. (B) Schematic configurations of the Ru(bpy)32+aptamer conjugates: I-The photosensitizer is linked to the 5'-end of the aptamer. II-The photosensitizer is linked to the 3'-end of the aptamer. III-The photosensitizer is linked to the 5'-

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end of the aptamer through a 4×thymidine (4×T) bridge. IV-The photosensitizer is linked to the 3'-end of the aptamer through a 4×thymidine (4×T) bridge.

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Figure 2. Stern-Volmer plots corresponding to the electron transfer quenching of the Ru(bpy)32+photosensitizer units by TA-MV2+: (a) Quenching of the conjugate I by TA-MV2+. (b) Quenching of the conjugate II by TA-MV2+. (c) Quenching of the conjugate III by TA-MV2+. (d) Quenching of the conjugate IV by TA-MV2+. (e) Quenching of the Ru(bpy)32+ linked to a nucleic acid consisting of a scrambled base sequence of the anti-tyrosinamide aptamer. (f) Quenching of the Ru(bpy)32+ separated from the anti-tyrosinamide aptamer in the presence of TA-MV2+.

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Figure 3. (A) Schematic photo-induced synthesis of NADPH driven by the different Ru(bpy)32+aptamer conjugates in the presence of TA-MV2+ and ferredoxin-NADP+ reductase (FNR), and appropriate control experiments. (B) Time-dependent absorption spectra corresponding to the photo-generated NADPH (λ = 340 nm) for different time-intervals by: Panel I-Conjugate IV; Panel II-Conjugate III; Panel III-Conjugate II; Panel IV-Conjugate I. Note that in all spectra the

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NADPH exists in equilibrium with TA-MV+• since FNR catalyzes the bi-directional transitions 2TA-MV+• + NADP+ + H+ ⇄ 2TA-MV2+ + NADPH. The NADPH is overexpressed in the equilibrated mixture and its relative content is controlled by the redox-potentials of the components. (C) Time-dependent concentrations of NADPH generated by: (a) Conjugate I, (b) Conjugate II, (c) Conjugate III, (d) Conjugate IV, (e) Ru(bpy)32+ linked to a nucleic acid consisting of a scrambled base sequence of the anti-tyrosinamide aptamer. (f) Ru(bpy)32+ separated from the anti-tyrosinamide aptamer. Error bars evaluated from N=3 experiments.

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Figure 4. (A) Schematic photo-induced evolution of hydrogen (H2) using the different Ru(bpy)32+-aptamer conjugates (and control systems) in the presence of TA-MV2+ and Pt nanoparticles as catalysts. (B) Time-dependent amounts of H2 generated by: (a) Conjugate I, (b) Conjugate II, (c) Conjugate III, (d) Conjugate IV, (e) Ru(bpy)32+ linked to a nucleic acid consisting of a scrambled base sequence of the anti-tyrosinamide aptamer. (f) Ru(bpy)32+ separated from the anti-tyrosinamide aptamer. Error bars evaluated from N=3 experiments.

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Figure 5. Transient decay curves corresponding to the quenching of the different Ru(bpy)32+aptamer conjugates, 25 μM, in the absence of TA-MV2+, curves (i), and in the presence of TAMV2+, 700 μM, curves (ii): A) Conjugate IV, B) Conjugate III, C) Conjugate II, D) Conjugate I.

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Table 1. Yield, Turnover Number and Quantum Yield corresponding to the synthesis of NADPH by the different Ru(bpy)32+-aptamer conjugates.[a] NADPH

Turnover

Quantum

(μM)a

Number

Yield

3' 4×T conjugate-IV

57

57

4.1%

5' 4×T conjugate-III

49

49

3.6%

3' conjugate-II

41

41

2.9%

5' conjugate-I

36

36

2.6%

Conjugate

[a]

Yield after an irradiation time-interval of 18 min, light intensity 20 mW.

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Table 2. Quantum yield corresponding to the photo-sensitized evolution of H2 in the presence of the different Ru(bpy)32+-aptamer conjugates. Conjugate

Quantum yield

3' 4×T conjugate-IV

3.9%

5' 4×T conjugate-III

3.2%

3' conjugate-II

2.2%

5' conjugate-I

1.8%

Scrambled

0.44%

Separated

0.31%

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Table 3. Dissociation constants of the Ru(bpy)32+-aptamer conjugates/TA-MV2+ and the static quenching rate constants corresponding to quenching of the different conjugates by TA-MV2+. Conjugate

Kd (nM)

ksq (108 s-1)

3' 4×T conjugate-IV

95±17

1.43±0.03

5' 4×T conjugate-III

84±20

1.25±0.05

3' conjugate-II

130±38

1.05±0.04

5' conjugate-I

140±47

0.87±0.03

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Table of Contents Graphic Artificial Photosynthesis with Electron Acceptor/Photosensitizer-Aptamer Conjugates

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