Photoinduced Cross-Linking of Short Furan ... - ACS Publications

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Photo-induced cross-linking of short furan-modified DNA on surfaces Cinthya Yamila Véliz Montes, Henry Memczak, Ellen Gyssels, Tomas Torres, Annemieke Madder, and Rudolf J. Schneider Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03855 • Publication Date (Web): 17 Jan 2017 Downloaded from http://pubs.acs.org on January 19, 2017

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Photo-induced cross-linking of short furan-modified DNA on surfaces Cinthya Véliz Montes†, ‡, Henry Memczak§, Ellen Gysselsǁ, Tomás Torres†,ǂ, Annemieke Madderǁ and Rudolf J. Schneider‡* †

BAM Federal Institute for Materials Research and Testing, Department of Analytical

Chemistry; Reference Materials, Richard-Willstätter-Str. 11, D-12489 Berlin, Germany. ‡

Department of Organic Chemistry, University Autónoma of Madrid, Cantoblanco, 28049

Madrid, Spain. §

IZI-BB Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and

Bioprocesses, Am Mühlenberg 11, D-14476 Potsdam, Germany. ǁ

Faculty of Sciences, Department of Organic and macromolecular Chemistry, Organic and

Biomimetic Chemistry Research Group, Ghent University, Krijgslaan 281 (S4), 9000 Ghent, Belgium. ǂIMDEA Nanoscience, c/Faraday,9, Cantoblanco, 28049 Madrid, Spain.

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ABSTRACT

We report for the first time the formation of site-specifically interstrand cross-linked (ICL) surface-immobilized furan-modified DNA duplexes via singlet oxygen. 1O2, necessary for effecting furan-mediated ICL formation, was produced in situ using methylene blue or a zinc phthalocyanine derivative (TT1) as photosensitizers. Via surface plasmon resonance spectroscopy we show that surface ICL was achieved and established a robust link which enhances the stability of the 12-mer duplex even after surface regeneration. The described method represents a novel platform technology based on surfaces with addressable and stable DNA duplex requiring only short oligonucleotides.

INTRODUCTION

DNA interstrand cross-link (ICL) formation has important clinical applications, as exploited in antitumor chemotherapeutics1-3. The development of methodologies for site-specific ICL formation is therefore an area of considerable interest. A new, furan-based ICL strategy has been widely studied in solution by Madder and co-workers4. The main advantage with respect to earlier developed approaches is the masked but inducible reactivity of furan-modified oligonucleotides that can selectively be activated by oxidation. Furan oxidation to a reactive keto-aldehyde leads to the fast formation of a covalent bond with the exocyclic amine functionality of adenine (A) or cytosine (C) bases of the complementary oligonucleotide sequence. This reaction results in the formation of a covalently linked and thus very stable DNA duplex that presents extraordinary resistance towards enzymatic digestion and higher melting temperatures in comparison with the non-cross-linked DNA duplex. Initial studies showed that, among the series of furan-modified nucleosides developed to date, the acyclic phenyl-furan-


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building block X (Table 1) offers a considerably high selectivity to form covalent bonds with complementary C and A bases, and upon cross-linking triggered by oxidation with Nbromosuccinimide (NBS) gives high cross-linking yield. Nevertheless, inspired by the mechanistic studies of the Vassilikogiannakis5 group on singlet oxygen-mediated furan oxidation in aqueous solution, a more biocompatible oxidation method based on the use of singlet oxygen, was developed and opens up the pathway to biological applications. Cross-linking efficiency with 1O2 versus NBS has been studied previously and the conditions to generate 1O2 were optimized in aqueous media allowing to obtain very high cross-linking efficiencies with methylene blue (MB) as photosensitizer (PS) and red light as excitation source4, 6. Other photosensitizers, e.g. phthalocyanines (Pc), that present high absorption and produce singlet oxygen in good quantum yields7, were also proposed as PSs for DNA cross-linking, although some obstacles had to be overcome8-11. DNA-directed immobilization (DDI) strategies provide high immobilization efficiency of DNA oligonucleotides (ODNs) on surface plasmon resonance (SPR) biosensor chips12-13. Analysis methods based on combining SPR and DDI allowed for the rapid determination of association and dissociation kinetics constants of various biomolecular complexes of interest14-15. In the present article, we demonstrate that covalent ICL of furan modified ODNs in solution can be applied also to surface-immobilized ODNs oligonucleotides using 1O2 allowing for the use of considerably shorter oligonucleotides than in classical hybridization-only based DDI. Additionally, the resulting DDI generated surfaces stable towards regeneration conditions. Proofof-principle is demonstrated by SPR measurements on a Biacore system using complementary oligonucleotide pairs for cross-linking and a fluorescein isothiocyanate (FITC)/anti-FITC probe as a model system in order to generate strong SPR signals16-17 as schematically illustrated in


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Figure 1. In addition to the SPR measurements, the high efficiency of the principle was confirmed by an Enzyme-linked Immunosorbent Assay (ELISA), which is described in the Supporting Information (Figure S1 and Figure S2).

Figure 1. Illustration of the method used in this work. First the gold chip was coated with neutravidin (NA) followed by the immobilization of the biotinylated and furan-modified capture ODN. The complementary FITC-modified ODN was allowed to hybridize to the capture ODN. Followed by the binding of an HRP-labelled anti-FITC antibody a) hybridized ODNs are efficiently separated after using Na2CO3 for regeneration, while b) oxidation of the furan using 1

O2 results in the formation of a rugged ICL, rendering the resulting short ODN duplex stable

towards the regeneration conditions. In this case, the detection antibody could still bind to the ODN duplex.

EXPERIMENTAL SECTION Instrumentation. The SPR measurements were performed using a Biacore™ T100 device (T200 Sensitivity Enhanced, GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and dextran-


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modified chips (G-COOH-sp) from Ssens (Enschede, The Netherlands). Experiments were performed at 25 °C using PBST (phosphate-buffered saline with 0.05% Tween20) as running buffer. Sensorgrams were double-referenced using an empty flow cell or a flow cell with noncomplementary DNA for subtraction of unspecific effects and buffer injections for correction of artefacts. The illuminator KL1500 LCD 150 W from Schott was used as cold red light source for irradiation. Chemicals. Methylene Blue (MB), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)N′-ethylcarbodiimide hydrochloride (EDC), and molecular biology grade buffer reagents were purchased from Sigma-Aldrich and used without further purification. Table 1 identifies the ODNs used in the experiments. The synthesis of the furan-modified building block (X) was performed as described previously18. Reagents for the synthesis of 5’biotin furan-modified ODNs were obtained from Glen Research. An ABI 394 DNA synthesizer was used for ODN synthesis and the products verified by mass spectrometry. The complementary 5’-FITC modified ODNs, streptavidin-coated 96-well microtiter plates and antiFITC-HRP-monoclonal antibody (Lot P319, 0.73 mg/mL) from mouse were purchased from BioTeZ (Berlin, Germany). The TT1 photosensitizer was prepared according to a method described previously10. Table 1. Oligonucleotide sequences and nomenclature DNA DNA X name sequence ODN1 ODN2 ODN3 ODN4 C.ODN1 C.ODN2 C.ODN3

5' biotin CTGACGGXGTGC 3' 5' biotin CAGTCGGXGAGC 3' 5' biotin GACTGCCXCACG 3' 5' biotin CACAGCCXCTCG 3' 3' GACTGCCCCACG-FITC 5' 3' GTCAGCCCCTCG-FITC 5' 3' CTGACGGCGTGC-FITC 5'


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C.ODN4

3' GTGTCGGCGAGC-FITC 5' FITC: Fluorescein isothiocyanate

General procedure for immobilization of biotinylated furan-modified ODNs. The gold chip was coated with neutravidin (NA) by EDC/NHS coupling. Unmodified reactive sites were then blocked with 1 M ethanolamine for 7 min. The respective biotinylated and furan-modified capture oligonucleotide (2 µM) was then injected for 2 min. Blocking of the NA reactive sites was performed by using 10 µM biotin for 1 min. All injections were carried out at a flow rate of 10 µL/min. Hybridization procedure. In order to obtain the association and dissociation curves of the DNA duplex, the complementary FITC-modified ODNs were allowed to hybridize to the capture ODNs by injecting different concentrations (0.01-10 µM) for 2 min at a flow rate of 30 µL/min. Dissociation of hybridized immobilized ODNs was achieved by injecting running buffer for 10 min at a continuous flow rate of 30 µL/min (Figure S3). Cross-linking experiments. Before taking the chip out of the SPR instrument for the ICL experiments, the binding of 48 nM HRP-labelled anti-FITC antibody to the hybridized FITCODN was performed, then 50 mM Na2CO3 was injected for 1 min as a test for the (in)stability of the DNA duplex. Hybridization and cross-linking were done in parallel by adding 100 µL of a mixture containing 1 µM FITC-ODN and the photosensitizer (5 µM MB or 5 µM TT1) to produce the necessary 1O2. Afterwards, the chip was irradiated with red light (658 nm, 8 cm distance, 5.2 mW/cm2) for 45 min while shaking at 350 rpm at room temperature. After washing with running buffer and drying of the chip, it was again mounted into the SPR instrument, where a first regeneration was carried out by 50 mM Na2CO3 for 1 min followed by the binding of 48


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nM HRP-labelled anti-FITC antibody. The same cross-link procedure was followed for the cross-link of the ODN2,3 and 4-C.ODN2,3 and 4 duplexes. The immobilized ODN1 was cross-linked by incubation with 1 µM C.ODN1 in PBS buffer and 5 µM MB. TT1 was initially dissolved in DMSO due to its low water solubility; the final concentration of TT1 was 5 µM. Covalent cross-linking in solution was proven using polyacrylamide gel electrophoresis and MALDI-TOF-MS (see Supporting Information), resulting in the observation of [M+H+]/2 at m/z 4,132.33 for ODN1-C.ODN1. Hybridization specificity experiments. Oligonucleotide sequences ODN2, 3 and 4 were immobilized in separate flow cells using the method described before. The hybridization specificity with complementary sequences C.ODN2, 3 and 4 were tested alone and in mixture. Kinetic data were obtained using five successive injections of recombinant GFP (Green Fluorescent Protein; single-cycle kinetics). Loading of the chip was calculated considering one resonance unit (RU) corresponding to one pg/mm² of bound biologic material19. Further data analysis of obtained sensorgrams was performed using Biacore™ T200 Evaluation Software v1.0 RESULTS AND DISCUSSION Exploiting a technique for covalent cross-linking of furan-modified ODNs in solution4 this is the first study to demonstrate this principle on flat surfaces by integration of three components: furan-modified short oligos (12-mers), oxidative coupling using a “residue-free” oxidant, singlet oxygen, and an efficient phthalocyanine photosensitizer functional with red light. Proof-ofprinciple is provided by the application to experiments on a chip surface followed by SPR and on a microplate surface as basis for an ELISA (described in the Supporting Information).


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The formation of a covalent bond between the immobilized ODN on a surface and the C.ODN target should enhance the stability of the duplex20. This duplex should be stable even in the presence of Na2CO3 which has a strong anion effect and stabilizes the single stranded conformation21. Using ODN1, DNA cross-linking on surface was investigated using 5 µM MB and a 5 µM Pc (TT1) at the conditions described above. While the yield of crosslinking in solution was determined to be around 28%4, first indications from SPR experiments, without excessive optimization of crosslinking conditions, indicate that the yield of DNA cross-link formation using methylene blue (MB) was 30-40 % and for the phthalocyanine TT1 ca. 5-10 %. As expected, the MB was more efficient in promoting ICL formation than the phthalocyanine TT1 (data not shown), whose lower photodynamic activity can result from the formation, to some extent, of molecular aggregates in water9. Figure 2 shows the SPR response obtained from the detection of ODN1 duplex by binding of the HRP-labelled anti-FITC antibody using 5 µM MB. When ODN1 was only hybridized (A1), thus allowing dehybridisation, the duplex was not stable to the regeneration step resulting in a decrease of the SPR response (A2). On the contrary, similar SPR response to the cross-linked ODNs after regeneration of the surface with Na2CO3 was observed (B1-B2). This is due to the increased stability of the ODN duplex by the cross-link formed. The somewhat lower signal (B1 vs. B2), about 20%, may be due to some singlet oxygen induced DNA damage. The anti-FITC antibody does not bind to the immobilized biotinylated and furan-modified capture oligonucleotide ODN1 (control), the antibody only recognizes the FITC from the respectively labelled sequence C.ODN1 once hybridization has occurred (Figure S4).


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three sequences yield highly specific binding; unspecific effects are < 2 %. Moreover, the specific detection of ODNs in solution was possible even for mixtures, laying the basis for a selective and stable platform formed by short ODN duplexes which can be used for multiplex bioanalytical applications.

Table 2. Binding signals in RU obtained by injection of C.ODNs 2, 3, & 4.

ODN2 ODN3 ODN4

2 351 5 1

3 1 324 2

C.ODNs 4 2+3 -10 331 -7 318 287 2

2+4 319 1 282

3+4 -3 292 284

2+3+4 327 308 288

CONCLUSIONS The present work shows that ICL formation of furan-modified DNA can be also implemented on a surface and is a very useful method to increase the stability of short immobilized DNA duplexes. SPR and ELISA experiments confirmed the ICL formation and the stability of the DNA after regeneration compared to the non-cross-linked ODN. One advantage of using shorter oligos is a reduction of the electrostatic barrier, formed by negatively charged ODNs, that otherwise can suppress the insertion of the complementary ODN in solution into the probe layer. Comparing photosensitizers, methylene blue (MB) is more efficient in promoting ICL formation than the phthalocyanine TT1. We have further demonstrated that it is possible to simultaneously use three different specific sequences for selective immobilization on a microarray surface. This technique represents a biomolecule-compatible immobilization method that could improve the robustness of array-based detection methods.


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ASSOCIATED CONTENT Supporting Information. Details of SPR and ELISA experiments. HPLC and PAGE runs and a MALDI-TOF-MS spectrum of ODN1-C.ODN1. 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 The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was funded by EU Marie Curie Actions, FP7-PEOPLE-2012-ITN, 316975, SO2S, and the Spanish MINECO (CTQ-2014-52869-P). E.G. is indebted to the Agency for Innovation by Science and Technology in Flanders (IWT). We thank Dr. Walter Stöcklein from Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB) for collaboration on the SPR measurements. ABBREVIATIONS ICL, DNA interstrand cross-link; NBS, N-bromosuccinimide; MB, methylene blue, Ps, photosensitizer; Pc, phthalocyanine; ODNs, oligonucleotides; SPR, surface plasmon resonance;


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ELISA, Enzyme-linked Immunosorbent Assay; HRP, horseradish peroxidase; FITC, Fluorescein isothiocyanate; NA, neutravidin; DDI, DNA-directed immobilization. REFERENCES 1. Rajski, S. R.; Williams, R. M., DNA cross-linking agents as antitumor drugs. Chem. Rev. 1998, 98 (8), 2723-2795. 2. Dronkert, M. L.; Kanaar, R., Repair of DNA interstrand cross-links. Mutat. Res. 2001, 486 (4), 217-47. 3. Huang, Y.; Li, L., DNA crosslinking damage and cancer - a tale of friend and foe. Transl. Cancer Res. 2013, 2 (3), 144-154. 4. Carrette, L. L. G.; Gyssels, E.; De Laet, N.; Madder, A., Furan Oxidation based Crosslinking: A New Approach for the Study and Targeting of Nucleic Acid and Protein Interactions. Chem. Commun. 2016, 52, 1539-1554. 5. Noutsias, D.; Alexopoulou, I.; Montagnon, T.; Vassilikogiannakis, G., Using water, light, air and spirulina to access a wide variety of polyoxygenated compounds. Green Chem. 2012, 14 (3), 601-604. 6. Schirmer, R. H.; Adler, H.; Pickhardt, M.; Mandelkow, E., "Lest we forget you-methylene blue...". Neurobiol. Aging 2011, 32 (12), 2325 e7-16. 7. Allen, C. M.; Sharman, W. M.; Van Lier, J. E., Current status of phthalocyanines in the photodynamic therapy of cancer. J. Porphyr. Phthalocya. 2001, 5 (2), 161-169. 8. Darwent, J. R.; Douglas, P.; Harriman, A.; Porter, G.; Richoux, M. C., Metal Phthalocyanines and Porphyrins as Photosensitizers for Reduction of Water to Hydrogen. Coord. Chem. Rev. 1982, 44 (1), 83-126. 9. Zhang, X. F.; Xu, H. J., Influence of Halogenation and Aggregation on Photosensitizing Properties of Zinc Phthalocyanine (ZnPC). Faraday Trans. 1993, 89 (18), 3347-3351. 10. Cid, J.-J.; Yum, J.-H.; Jang, S.-R.; Nazeeruddin, M. K.; Ferrero, E. M.; Palomares, E.; Ko, J.; Graetzel, M.; Torres, T., Molecular cosensitization for efficient panchromatic dyesensitized solar cells. Angew. Chem. Int. Ed. 2007, 46 (44), 8358-8362. 11. Contreras, L. E. S.; Zirzlmeier, J.; Kirner, S. V.; Setaro, F.; Martinez, F.; Lozada, S.; Escobar, P.; Hahn, U.; Guldi, D. M.; Torres, T., Cholesteryl oleate-appended phthalocyanines as potential photosensitizers in the treatment of leishmaniasis. J. Porphyr. Phthalocya. 2015, 19 (13), 320-328. 12. Niemeyer, C. M.; Boldt, L.; Ceyhan, B.; Blohm, D., DNA-Directed immobilization: efficient, reversible, and site-selective surface binding of proteins by means of covalent DNAstreptavidin conjugates. Anal. Biochem. 1999, 268 (1), 54-63. 13. Vigneshvar, S.; Sudhakumari, C. C.; Senthilkumaran, B.; Prakash, H., Recent Advances in Biosensor Technology for Potential Applications – An Overview. Frontiers Bioeng. Biotechnol. 2016, 4, 11. 14. Hu, W.-P.; Huang, L.-Y.; Kuo, T.-C.; Hu, W.-W.; Chang, Y.; Chen, C.-S.; Chen, H.-C.; Chen, W.-Y., Optimization of DNA-directed immobilization on mixed oligo(ethylene glycol) monolayers for immunodetection. Anal. Biochem. 2012, 423 (1), 26-35. 15. Nguyen, H. H.; Park, J.; Kang, S.; Kim, M., Surface plasmon resonance: a versatile technique for biosensor applications. Sensors 2015, 15 (5), 10481-510. 16. Schenk, J. A.; Sellrie, F.; Böttger, V.; Menning, A.; Stöcklein, W. F.; Micheel, B., Generation and application of a fluorescein-specific single chain antibody. Biochimie 2007, 89 (11), 1304-11.


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17. Hovestädt, M.; Memczak, H.; Pleiner, D.; Zhang, X.; Rappich, J.; Bier, F. F.; Stöcklein, W. F., Characterization of a new maleimido functionalization of gold for surface plasmon resonance spectroscopy. J. Mol. Recognit. 2014, 27 (12), 707-13. 18. Stevens, K.; Claeys, D. D.; Catak, S.; Figaroli, S.; Hocek, M.; Tromp, J. M.; Schurch, S.; Van Speybroeck, V.; Madder, A., Furan-Oxidation-Triggered Inducible DNA Cross-Linking: Acyclic Versus Cyclic Furan-Containing Building Blocks-On the Benefit of Restoring the Cyclic Sugar Backbone. Chem. - Eur. J. 2011, 17 (25), 6940-6953. 19. Karlsson, R.; Fagerstam, L.; Nilshans, H.; Persson, B., Analysis of active antibody concentration. Separation of affinity and concentration parameters. J. Immunol. Methods 1993, 166 (1), 75-84. 20. Xue, X.; Xu, W.; Wang, F.; Liu, X., Multiplex Single-Nucleotide Polymorphism Typing by Nanoparticle-Coupled DNA-Templated Reactions. J. Am. Chem. Soc. 2009, 131 (33), 1166811669. 21. Galyuk, E. N.; Lando, D. Y.; Egorova, V. P.; Dai, H.; Dosin, Y. M., Na2CO3 influence on DNA double helix stability: Strong anion destabilizing effect. J. Biomol. Struct. Dyn. 2003, 20 (6), 801-809.


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