Addressable DNA-Myoglobin Photocatalysis - Wiley Online Library

FULL PAPERS DOI: 10.1002/asia.200900082

Addressable DNA–Myoglobin Photocatalysis Chi-Hsien Kuo,[a] Ljiljana Fruk,[a, b] and Christof M. Niemeyer*[a]

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2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Abstract: Photoactivatable myoglobin containing a DNA oligonucleotide as a structural anchor was designed by using the reconstitution of artificial heme moieties containing Ru3 + ions. This semisynthetic DNA–enzyme conjugate was successfully used for the oxidation of peroxidase substrates by

using visible light instead of H2O2 for the activation. The DNA anchor was utilized for the immobilization of the Keywords: bioorganic chemistry · DNA · photochemistry · protein conjugates · redox chemistry

Introduction The design of novel biocatalysts is of paramount interest for applications in synthetic bio-organic chemistry and biosensing. A promising route to novel catalysts takes advantage of naturally occurring enzymes, which are modified with synthetic groups to alter existing and create novel catalytic reactivities. For example, semisynthetic metalloenzymes can be designed and engineered to contain non-native metalcontaining cofactors.[1–3] In particular, synthetic analogues of the metalloenzymes native prosthetic group are introduced into apoenzymes, which are the native enzymes lacking their natural prosthetic groups, to change, fine tune, or even establish entirely new catalytic specificities. To this end, several successful approaches on the design of semisynthetic metalloenzymes, containing heme (protoporphyrin IX) as prosthetic group, have been reported by Hayashi and co-workers,[4] Shinkai and co-workers,[5–7] and others.[8] A particular challenging goal concerns the design of photoactivatable hybrid enzymes, because activation by light would provide not only temporal and spatial control over enzyme activity but it could also eliminate the need for oxidative activators, such as H2O2 for peroxidases or NAD(P)H for oxygenases. To this end, native apoenzymes have been reconstituted

[a] C.-H. Kuo, Dr. L. Fruk, Prof. Dr. C. M. Niemeyer Technische Universitt Dortmund Fakultt Chemie Biologisch-Chemische Mikrostrukturtechnik Otto-Hahn Str. 6, 44227 Dortmund (Germany) Fax: (+ 49) 231-755-7082 E-mail: [email protected] [b] Dr. L. Fruk Centre for Functional Nanostructures Universitt Karlsruhe Wolfgang Gaede Str. 1 76131 Karlsruhe, Germany

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enzyme on the surface of magnetic microbeads. Enzyme activity measurements not only indicated undisturbed biofunctionality of the tethered DNA but also enabled magnetic separationbased enrichment and recycling of the photoactivatable biocatalyst.

with heme derivatives, bearing photoactive groups, which can harvest light, harness the energy for redox reactions, and transfer generated electrons to the heme center to enable downstream redox reactions of the enzyme. As an example, Hamachi et al. reported on a heme derivative that contained the photosensitive tris(2,2’-bipyridyl)ruthenium(II) (RuACHTUNGRE(bpy)32 + moiety. Insertion of this group into apomyoglobin (aMb) led to a hybrid enzyme that could be effectively activated by visible light.[6, 7] Herein, we report on the installment of this approach towards the synthesis of a next generation of hybrid biocatalysts that can be site-selectively immobilized on solid supports. We have previously used reconstitution of apoenzymes for the synthesis of semi-synthetic DNA oligonucleotide conjugates of myoglobin (Mb)[9, 10] or horseraddish peroxidase (HRP).[10–12] Owing to the highly selective binding capabilities of the DNA oligomers, these conjugates can be specifically immobilized at solid surfaces by means of DNA hybridization. Thus, they can be considered as programmable catalysts that are useful for applications in biocatalysis or sensing.[11] Figure 1 shows our synthesis of heme derivative 4, containing a single-stranded DNA oligonucleotide and a photosensitive tris(2,2’-bipyridyl)ruthenium(II) (RuACHTUNGRE(bpy)32 + moiety. Heme 4 was then used for reconstitution of apo-myoglobin (apoMb) to yield the hybrid enzyme Mb-4 (Figure 1 b). The modified enzyme was investigated for its light-driven peroxidase activity by using the chloropentamminecobaltACHTUNGRE(III) complex ([CoACHTUNGRE(NH3)5Cl]2 + ) as the sacrificial electron acceptor and 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS) as the peroxidase substrate. We observed that Mb-4 showed significant enzymatic activity and demonstrated that such hybrid enzymes can be site-specifically immobilized on complementary DNA oligonucleotides that are bound to the surface of magnetic microbeads.


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Figure 1. Schematic representation of the synthesis of hybrid myoglobin (Mb) containing the artificial cofactor 4, which contains both a singlestranded oligonucleotide and a photosensitive tris(2,2’ bipyridyl)ruthenium(II) (3). As shown in (b), cofactor 4 was used for reconstitution of apoMb.

Results and Discussion Synthesis of Heme Conjugate 4 and Reconstitution of Apoenzymes [7]

Inspired by the work of Hamachi et al., we developed a synthetic route for the synthesis of heme derivative 4, which contains both a single-stranded oligonucleotide and the photosensitive tris(2,2’-bipyridyl)ruthenium(II) (RuACHTUNGRE(bpy)32 + moiety. As shown in Figure 1 a, 5’-alkylamino-modified oligonucleodide 1, which remained immobilized on the controlled-pore glass (CPG) support after phosphoramidite oligonucleotide synthesis, was coupled to heme through its propionate side chains by using HBTU/HOBt/DIPEA (HBTU = O-benzotriazole-N,N,N,N-tetramethyl-uroniumhexafluorophosphate, HOBt = 1-hydroxybenzotriazole, DIPEA = diisopropylethylamine) as activating reagents for amide synthesis.[9] As previously investigated, two products are formed by this reaction that are heme derivatives and which contain either one or two DNA oligomers (hemeD1 and hemeD2, respectively, 2 in Figure 1 a) linked to the propionate chains. The CPG column was washed and the products were then reacted with an excess of alkylamino-modified tris(2,2’-bipyridyl)ruthenium(II) (RuACHTUNGRE(bpy)32 + ion (3 in Figure 1), which was synthesized according to previously reported procedures,[7] to yield heme derivative 4 (see Figure S1 in the Supporting Information).[13] Subsequent to the cleavage of 4 from the CPG support by overnight treatment with ammonium hydroxide, the crude reaction mixture was analyzed and purified by HPLC chromatography (Figure 2 a). From the chromatogram it was evident that in addi-

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Figure 2. Chromatograms of HPLC analysis of cofactor 4 (a) and FPLC purification of Mb-4 (b). Unreacted amino-modified DNA is marked 1, HemeD2 corresponds to heme linked to two DNA strands, which was not used further in this study, and peak 4 is the heme–Ru–DNA cofactor.

tion to the unmodified aminoalkyl–oligonucleotide (peak 1 in Figure 2 a) and formation of hemeD2 (peak 2 in Figure 2 a), the desired Ru-modified heme 4 was formed (peak 3 in Figure 2 a), in approximately 35 % yield with respect to the used amount of DNA.[13] The fractions were collected and successful synthesis of 4 was confirmed by MALDI-MS analysis (see Figure S2 in the Supporting Information). Subsequently, 4 was used for the reconstitution of apoMb, as schematically depicted in Figure 1 b, and the resulting reaction mixture was analyzed by fast protein liquid chromatography (FPLC; Figure 2 b). The formation of reconstituted hybrid enzyme Mb-4 was indicated by the presence of a peak with a retention time of about 350 s, baseline separated from excess educt 4 (Figure 2 b). The successful formation of the reconstituted enzymes was also verified by gel electrophoretic analyses (see Figure S3 in the Supporting Information). These gels indicated the formation of conjugate species with increased electrophoretic mobility, as compared with the native Mb, owing to the appended negatively charged DNA oligonucleotide.


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Photocatalysis of DNA–Myoglobin

Light-Driven Peroxidase Activity of Mb-4 With the semisynthetic enzyme Mb-4 in hand, we initially investigated its catalytic activity in a light-driven peroxidation reaction (Figure 3). To this end, we employed 2,2’azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS), a typical substrate for peroxidase enzymes that is oxidized to form colored radical products that are detectable photospectrometrically at an absorption wavelength of 405 nm.[14] ABTS had also been used in the previous studies by Hamachi et al.[7] on semisynthetic Mb, reconstituted with a RuACHTUNGRE(bpy)32 + –heme moiety. The photoinduced peroxidation requires the presence of [CoACHTUNGRE(NH3)5Cl]2 + ion, which acts as an electron acceptor. It has previously been used for this purpose and the detailed mechanism of this multi-step reaction

has been proposed earlier[7] (see also Figure S4 in the Supporting Information). Here, we initially investigated whether the DNA-containing enzyme Mb-4 is also capable of catalyzing this photochemical peroxidation. Indeed, the results shown in Figure 3 clearly indicated that hybrid Mb-4 can act as a photocatalyst. The activity of Mb-4 in the light-induced peroxidation was compared with that of the native enzyme (Mb in Figure 3) in both the light-induced and H2O2-mediated reaction. It was evident that the Mb-4 was only slightly less active in the photoreaction, as compared with native Mb in the conventional H2O2-triggered reaction. As expected, neither the native enzymes nor native heme or 4 showed any activity in the photoreaction.

Figure 3. Light-triggered peroxidation of ABTS in the presence of a sacrificial electron-acceptor [CoACHTUNGRE(NH3)5Cl]2 + ion, catalyzed by hybrid enzyme Mb-4. The bars in (b) represent absorbance values of the colored ABTS radical product, which were determined spectrophotometrically at l = 405 nm in endpoint measurements. Enzyme (100 pmol) was used in the reactions and controls of the H2O2-mediated reaction (left set of bars) was carried out with 400 mm H2O2 in the absence of the [CoACHTUNGRE(NH3)5Cl]2 + ion.

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DNA-Directed Immobilization To investigate whether the DNA moiety of hybrid Mb-4 is still functional, we carried out immobilization experiments on magnetic microbeads, functionalized with complementary oligonucleotides (Figure 4). To this end, two Mb-4 hybrids were synthesized (Mb-4 a, Mb4 b) by reconstitution of apoMb by using Ru-/DNAmodified heme cofactors (4 a, 4 b) that contained oligonucleotides of different sequences. The two hybrid enzymes were allowed to bind to two batches of microbeads (beads Bca and BcB), containing capture oligonucleotides, which were complementary to either Mb4 a (beads Bca, in Figure 4 b) or Mb-4 b (beads Bcb, in Figure 4 B). Owing to the lower amounts of enzymes (approximately 40 pmol) attached to the microbeads, it was necessary to use the fluorogenic substrate Amplex Red instead of ABTS, which allows for improved sensitivity in the detection of peroxidase activity owing to the formation of highly fluorescent resorufin (Figure 4 a). Initially, the peroxidase activity of Mb-4 was tested in the H2O2-mediated reaction (Figure 4 b). It was clearly evident that resorufin formation occurred only in the case of complementary pairs of

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idase reactivity with this substrate; however, signals were less than 30 % of those obtained for the respective enzymes. The lack of peroxidase activity of 4 in the ABTS measurements (Figure 3) can be explained by the increased sensitivity of fluorescence detection of resorufin. Specific hybridization of Mb-4 with the bead-bound capture oligonucleotides was also evident in the light-driven peroxidation (Figure 4 c). There, the presence of the [CoACHTUNGRE(NH3)5Cl]2 + ion led to strongly increased background values owing to the direct oxidation of Amplex Red by Co3 + ions. Subtraction of background values, however, indicated the same results as in the H2O2mediated reaction, that is, that cofactors 4 posses peroxidase activity and immobilization occurred owing to specific Watson–Crick base pairing. We also investigated whether the DNA moiety in Mb-4 remained intact and functional during the light-driven peroxidation in the presence of [CoACHTUNGRE(NH3)5Cl]2 + . To this end, we used the capture oligonucleotide-modified magnetic microbeads to extract Mb-4 from a reaction solution after the photoinduced reaction was performed (see Figure S5 in the Supporting Information). Magnetic separation, followed by washing and renewed addition of the peroxidase substrate and [CoACHTUNGRE(NH3)5Cl]2 + ion clearly indicated the presence of active catalyst on the microbeads. To demonstrate the recyclability of this hybrid catalyst, a set of experiments was repeatedly carried out with the same batch of the bead-immobilized enzyme (see Figure S5 b in the Supporting Information). It was observed that the catalytic efficiency of the immobilized enzyme remained largely preserved, however, it decreased with the number of consecutive experiments to about 50 % of the original activity after the 6th repetition. We attribute this loss in activity to loss of the enzyme from the beads owing to extensive washing. These results thus proved not only that Mb-4 did retain its capability of nucleic acid hybridization during photocatalyis, but the experiments also demonstrated that the catalyst can be readily recycled from complex reaction mixtures.

Conclusions

Figure 4. DNA-directed immobilization of 4 and Mb-4 on microbeads containing complementary capture oligonucleotides. The procedure is schematically depicted in (a): two different conjugates (4 a, 4 b, or Mb-4 a, Mb-4 b) containing oligonucleotides of different sequence were allowed to bind to two batches of beads containing capture oligomers that are complementary to either 4 a, Mb-4 a (beads Bca) or 4 b, Mb-4 b (beads Bcb), respectively. Fluorogenic dye Amplex Red was used as the substrate, which was oxidized to highly fluorescent resorufin. The bars in (b) represent endpoint measurements of signals generated in the H2O2-mediated reaction, whereas the bars in (c) represent background-corrected signals measured in the light-driven peroxidation in the presence of the sacrificial electron-acceptor [CoACHTUNGRE(NH3)5Cl]2 + ion.

Herein, we have demonstrated that the reconstitution of apo-myoglobin can be used to prepare semisynthetic enzymes that contain a Ru3 + moiety for photoactivation in addition to a single-stranded DNA moiety for specific hybridization-based immobilization. The resulting hybrid enzyme was capable of both catalyzing light-induced peroxidation reactions and specific nucleic acid hybridization. The latter allowed us to selectively immobilize the catalyst to solid supports and also opened up a means for catalyst recycling by magnetic separation. We anticipate that this principle can be applied to a range of heme-dependent enzymes, thus enabling the generation of novel classes of chemically addressable, light-triggered photocatalysts.

enzyme- and bead-attached oligonucleotides. Interestingly, the Ru/DNA-modified heme cofactors also revealed perox-

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Photocatalysis of DNA–Myoglobin

Experimental Section Ruthenium ligand 3: The RuACHTUNGRE(bpy)32 + complex 3 was synthesized by adopting the approach of Hamachi and co-workers.[6, 7] Detailed experimental procedures are provided in the Supporting Information. Synthesis of modified heme 4 a and 4 b: Amino modified oligonucleotides (Da = 5’-trityl-amino-GTG GAA AGT GGC AAT CGT GAA G and Db = 5’-trityl-amino-GGA CGA ATA CAA AGG CTA CAC G), which contained the protection groups and were still coupled to the CPG support, were purchased from SIGMA. The trityl group was removed by using commercial 3 % dichloroacetic acid (DCA) in dichloromethane solution followed by washing with CH3CN and drying with argon. Hemin coupling was carried out as described previously by using a HBTU, HObt coupling procedure.[9, 10] The modified oligonucleotides were deprotected by using a tert-butylamine/MeOH/HO (1:2:1) mixture for 3 h at 65 8C and purified by HPLC.[9, 10] Myoglobin reconstitution: Apo Mb (aMb) was prepared by Teales 2-butanone method.[15] In brief, a solution of the apo-enzyme (200 mL, 60 mm) in potassium phosphate buffer solution, pH 7, was mixed with 4 (1.1 eq, 330 mL, 40 mm) and incubated for 24 h at 4 8C. Reconstituted enzyme were purified by using ion-exchange FPLC (AKTA purifier, Amersham Bioscience, MonoQ column, buffer A solution: 20 mm Tris A pH 8.3 and buffer B solution: 20 mm Tris A and 1.5 m NaCl using a stepwise gradient; Tris = tris(hydroxymethyl)aminomethane). The concentration was determined spectrophotometrically. Enzymatic reactions: Co–KPi buffer solution (KPi = postassium phosphate) was prepared by adding [CoIIIACHTUNGRE(NH3)5Cl]Cl2 to 50 mm phosphate buffer solution, such that the final concentration of [CoIIIACHTUNGRE(NH3)5Cl]Cl2 was 1.0 mm. The pH of the buffer solution was adjusted to 8.8 and oxygen was removed by argon bubbling for 30 min. Photo-irradiation of the solutions was carried out for 30 min at room temperature by using a 250 W high-pressure Hg lamp equipped with an optical filter (l > 450 nm). 100 pmol enzyme (0.67 mm in Co-KPi buffer) and 5 nmol ABTS (33 mm in Co–KPi buffer) or AmplexRed (33 mm in Co–KPi buffer) were used in the enzymatic assays. Oxidation of ABTS was monitored by absorbance measurements at 405 nm, which corresponds to the production of cationic radicals. Amplex Red conversion was followed by fluorescence measurements (excitation 530 nm, emission 590 nm). Spectral changes were monitored by using a Synergy HT microplate reader from BioTek (running Software KC4 Version 3.4 Rev. 12). DNA-directed immobilization (DDI) on magnetic beads: The streptavidin-coated magnetic bead (MB) suspension (Invitrogen Dynabeads MyOne Streptavidine C1) was thoroughly mixed for at least 30 seconds prior to each modification step. For each sample vial, 100 mL of suspension was taken from the stock and washed three times with TETBS buffer solution (200 mL). Biotinylated single-stranded DNA (1000 pmol cDa or cDb; 10 mL, 100 mm) was then added to the bead suspension and incubated for at least 2 h at room temperature. The suspension was washed three times with biotin–TETBS buffer solution (200 mL), followed by threefold washing with phosphate-buffered saline solution. Cofactor (150 pmol) or reconstituted enzyme (1.0 mm in Co–KPi buffer solution) were added to the vial containing the MB–single-stranded DNA suspension and incubated at room temperature under orbital shaking overnight. The resulting suspension was then washed with TETBS buffer (3 200 mL) followed by threefold washing with the reaction buffer (KPi for H2O2 reaction and Co–KPi for light irradiation reaction) and kept in the reaction buffer at 4 8C for further use. After each wash, the magnetic beads were removed from the solution by using a magnetic separator (Biometra LABOR 1600).

Acknowledgements This work was supported by the Zentrum fr Angewandte Chemische Genomik (ZACG), a joint research initiative founded by the European Union, and the Ministry of Innovation and Research of the state Northrhine Westfalia. CMN thanks the Max-Planck Society for financial support of a Max-Planck Fellow research group at the Max Planck Institute of Molecular Physiology, Dortmund. LF was supported by Marie Curie

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International Incoming Fellowship (project 514582). C.-H.K. acknowledges support through the International Max-Planck Research School in Chemical Biology, Dortmund and a student fellowship from Deutscher Akademischer Austauschdienst (DAAD). The authors wish to thank Filip and Franjo Kis for the design of the frontispiece.

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Received: February 25, 2009

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