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Short Technical Reports amounts of methylated cytosine. Fungal Genet. Biol. 22:103-111. 8.Kerr, S.E., K. Seraidarian and M. Wargon. 1949. Studies on ribonucleic acid: II. Methods of analysis. J. Biol. Chem. 181:761-771. 9.Linsmaier, E.M. and F. Skoog. 1965. Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18:100-127. 10.Loring, H.S., J.L. Fairley, H.W. Bortner and H.L. Seagran. 1952. A spectrophotometric method for the analysis of the purine and pyrimidine components of ribonucleic acid. J. Biol. Chem. 197:809-821. 11.Raizis, A.M., F. Schmitt and J.P. Jost. 1995. A bisulfite method of 5-methylcytosine mapping that minimizes template degradation. Anal. Biochem. 226:161-166. 12.Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., p. 9.34. CSH Laboratory Press, Cold Spring Harbor, NY. 13.Schmidt, G. and P.A. Levene. 1938. Ribonucleodepolymerase (The Jones-Dubos enzyme). J. Biol. Chem. 126:423-434. 14.Schmitt, F., E.J. Oakeley and J.P. Jost. 1997. Antibiotics induce genome-wide hypermethylation in cultured Nicotiana tabacum plants. J. Biol. Chem. 272:1534-1540. 15.Shabarova, Z. and A. Bogdanov. 1994. Advanced Organic Chemistry of Nucleic Acids, p. 60. VCH Verlagsgesellschaft mbH, Weinheim, Germany. 16.Vögeli-Lange, R., C. Fründt, C.M. Hart, R. Beffa, F. Nagy and F. Meins, Jr. 1994. Evidence for a role of ß-1,3-glucanase in dicot seed germination. Plant J. 5:273–278. 17.Woodcock, D.M., P.J. Crowther, D.L. Simmons and L.A. Cooper. 1984. Sequence specificity of cytosine methylation in the DNA of the Chinese hamster ovary (CHO-K1) cell line. Biochim. Biophys. Acta 783:227233. 18.Wu, J., J.P. Issa, J. Herman, D.E. Bassett, Jr., B.D. Nelkin and S.B. Baylin. 1993. Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells. Proc. Natl. Acad. Sci. USA 90:8891-8895.

We would like to thank Dr. J. Hofsteenge’s group for allowing us to use their UV/vis spectrophotometer and luminescence spectrometer, without which this work would have been impossible. We would also like to thank the group of Dr. A. Matus for useful discussions and helping us resolve many of the technical problems we encountered during the development of this protocol. We also thank Dr. J. Hofsteenge and Prof. W. Filipowicz for their critical reading of this manuscript. Address correspondence to Dr. Jean-Pierre Jost, Friedrich Miescher Institute, Postfach 2543, CH-4002 Basel, Switzerland. Internet: [email protected]

Edward J. Oakeley, Frédéric Schmitt1 and Jean-Pierre Jost2 University of Exeter Exeter, England, UK 1Rhône-Poulenc Agro Lyon, France 2Friedrich Miescher Institute Basel, Switzerland

Changing Functionality of Surfaces by Directed Self-Assembly Using Oligonucleotides—The Oligo-Tag BioTechniques 27:752-760 (October 1999)

ABSTRACT A method is presented to modify surfaces for biotechnological applications. Oligonucleotides have been coupled covalently to a pre-activated surface. Complementary oligonucleotides hybridize to the surface, which are conjugated with functionalities. The oligonucleotides serve as “Oligo-Tags” for these functionalities that now are linked specifically and reversibly. The approach might be used to change DNA-arrays into arrays of arbitrary ligands. We demonstrate the method with an optical wave guide grating coupler as a sensing surface using two different haptens as examples for a variety of functionalities. The haptens were 2,4-dichlorophenoxyacetic acid and atrazin and are recognized by specific antibodies. The surface created was completely regenerable by alkaline washing or temperature increase without any loss of binding capacity. Specificity was demonstrated by competitive binding of antibody in presence and absence of analyte; unspecific binding has not been observed.

INTRODUCTION Received 20 July 1998; accepted 26 May 1999. 752 BioTechniques

The highly ordered microstructure

of the living world serves as a guide for either the construction of biomimetic devices or by giving access to the direct use of biomolecules. In biotechnology, it is often necessary to couple the biomolecule to a solid surface. Such a surface might be a silicium wafer, a metal or organic polymer or any kind of glass, as is used in this paper. Several methods have been developed to couple biomolecules to solid supports for a variety of applications (1,15,19). Most of these methods link the molecules irreversibly to the surface, e.g., by physisorption or by covalent coupling of receptors to an activated surface. As a consequence of such methods, the molecules are statistically distributed and often only poorly oriented. Active biomolecules, binders as well as catalysts, are highly flexible molecules with a limited lifetime in their natural environment. The living cell overcomes this problem by continuous exchange of functional molecules, i.e., destruction and synthesis of active macromolecules. The lifetime of devices relying on biomolecules is restricted by the lifetime of the isolated biomolecule. Attempts to stabilize the molecules and restore their functionality have been only partially successful (21). What we can learn from the living cell for biotechnological in vitro applications is to exchange the biomolecule without changing the device. In this report, we introduce a method for directed self-assembly of defined surface coverage by the use of oligonucleotides, making use of the high specificity of the double-stranded formation of natural nucleic acids. Two oligonucleotides form a strong dimer by hydrogen bonds called hybrids when both strands are complementary. The strength of the dimerization depends on (i) the number of matching bases in a sequence, (ii) the number and loci of mismatches and (iii) the base composition. Double-strand stability is well investigated and applied in all types of gene probe analysis, which is hybridization-based (20). To separate the two complementary strands, destabilizing conditions have been proven in numerous applications such as high temperature, high ionic strength or alkaline environment (6). Separability is given for all kinds of homo- and hetVol. 27, No. 4 (1999)

Short Technical Reports erodimers including natural DNA, RNA and any stabilizing modification thereof, as well as for the recently synthesized peptide nucleic acids (PNA) (16) and other nucleotide mimics like phosphoric acid ester nucleic acid (PHONA) (18). These features of oligonucleotides make them ideal candidates for creating reversible surface modifications by serving as linkers between the surface and a functional molecule. An example of this kind has been given by Niemeyer et al., who used streptavidin-biotin as a second bridge (17). The authors also demonstrated the possibility of using the streptavidin-biotin bridge to switch from nucleic acid arrays to protein arrays. The oligomer as a tag for a functional molecule has the advantage of being very specific. Thus, the tagged molecule can be guided by means of the sequence. The principle of the method becomes “visible” when the functional modifications are molecular recognition ele-

ments, which can be transduced by an appropriate sensing device. We demonstrate the method with the example of two different immunoassays that are performed on the same prepared sensing surface. However, we point out that this type of application is for visibility only and that the basic notion can easily be transferred to other applications (e.g., linkage of enzymes). MATERIALS AND METHODS The sequences of the oligomers were chosen from plasmid pBR322 only, with equal amounts of G, C, A and T: Oligomer 1, p-ATC TAC CTG

CCT GGA CA; Oligomer 2, 2,4-DTGT CCA GGC AGG TA; Oligomer 3, AHA-TGT CCA GGC AGG TA; Oligomer 4, TGT CCA GGC AGG TA. The oligomers were synthesized by BioTeZ (Berlin-Buch, Germany), and 2,4-dichlorophenoxyacetic acid (2,4D), 2-ethylamino-4-chloro-6-isopropylamino-1,3,5-triazin (atrazin) and 2-aminohexanoic-acid-4-chloro-6-isopropylamino-1,3,5-triazin (AHA) were from Riedel-de-Haën (Seelze, Germany). For coupling, Oligomer 4 was activated at the 5′ end by an aminohexylgroup and purified by a Sephadex® G25 column (Amersham Pharmacia Biotech, Uppsala, Sweden); 2,4-D and AHA were dissolved in dimethylfor-

Figure 1. Scheme of the method for changing functionality of a surface by oligonucleotide-tagged functionalities. Vol. 27, No. 4 (1999)

A

B

Figure 2. (A) Sensorgram of a real time measurement of hybridization and antibody binding using a grating coupler. The sensor surface is loaded by Oligomer 2, a complementary 14-mer conjugated with AHA at the 5′ end (a). Incubation of Oligomer 2 was stopped by rinsing buffer (a′). Anti-atrazin-antibody binding to the modified surface starts at (b) and ends at (b′). At (c), regeneration with 50 mM NaOH is started, and the recovered surface is at the starting level at (c′). (B) A complete sequence of binding and inhibition of different oligonucleotides and antibodies as given in the scheme of Figure 1. See text for details.

mamide (20 mg/mL) and activated using N-hydroxysuccinimide. The activated Oligomer 4 was dissolved in sodium carbonate buffer (0.1 M, pH 9.0), and mixed 1:1 vol/vol with the activated 2,4-D and AHA, respectively, and incubated overnight at room temperature. The modified oligomers were purified by reverse-phase HPLC. The 5′ end phosphorylated Oligomer 1 was activated using 1-methylimidazol and N-(3-dimethyl-aminopropyl) -N′-ethyl-carbodiimide-hydrochloride (EDC) as previously described (14). The coupling procedure follows in brief: The sensor chip was cleaned by sulfuric acid including 10% hydrogen-superoxide, washed in aqua dest and immersed for 5 min in 5 M NaOH. After consecutively removing H2O with dry acetone (3×), the sensor chip was incubated overnight with 100 mM carbonyldiimidazole (CDI) in dry acetone. The activated sensor chip was washed again with acetone and air-dried. The sensor chip was incubated in 100 mM diaminoethane in phosphate buffer (50 mM, pH 6.0) for 4 h. After multiple alternative washings in phosphate solutions (50 mM) of pH 3.0 and 8.0, the sensor chip was incubated in EDC/DNA solution overnight and rinsed again in the two different phosphate solutions. The sensor chips were used immediateVol. 27, No. 4 (1999)

ly after preparation. From other experiments, we know that they may be stored at room temperature for more than 6 months without loss of binding capacity. Grating couplers, originally produced for communications industries, are surface-sensitive devices that can be used for surface-bound macromolecular interaction analysis. Refractive index changes in the vicinity of the surface of a thin planar light wave guide are reflected in changes of the in-coupling angle of a guided mode (22). The measuring system used here obtains the loss of energy of the reflected light by the in-coupling mode (4) similar to surface plasmon resonance (SPR) devices (13). Grating coupler chips ASI-3200 were obtained from Artificial Sensing Instruments (Zürich, Switzerland). The grating coupler measuring device was connected to a simple flowthrough system that forces the sample through the measuring cell (3). The running buffer in all experiments was 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA. Antibodies were diluted in running buffer as indicated and incubated with the analyte 30 min before incubation. Antibody against atrazin was the monoclonal K4E7 from Giersch and Hock (8), the monoclonal antibody E2/G2 against 2,4-D was from Fránek et al. (7). BioTechniques 755

Short Technical Reports RESULTS AND DISCUSSION As the sensing element, a grating coupler (22), which measures refractive index changes in the near vicinity of a surface by means of the evanescent field, has been chosen. In our experiments, this sensing device serves as a model for any kind of surface-sensitive device. It might be substituted, e.g., by a metallic film for SPR or piezoelectric transducers. For grating couplers, the surface loading by adsorption or binding is proportional to the refractive index change, if the refractive index of the bound species is higher than that of the buffer solution (nwater = 1.33, nprotein = nDNA = 1.56). Thus, binding of macromolecules onto the sensor surface gives a positive signal, while release of bound molecules leads to a decrease of the signal. The primary signal, change of refractive index, may be recalculated to the surface loading of bound molecules given in ng/mm2 (22). Following the time course of such a signal is sometimes called “sensorgram” (Figure 2). The principle of the idea is sketched in Figure 1. A desoxyriboseoligonucleotide, Oligomer 1, of defined length and sequence is covalently coupled at the 5′ end to a sensing surface. Oligomer 2, which has the complementary sequence, was linked to a derivative of an analyte (A), and hybridized to the surface coupled Oligomer 1 (Figure 1, 1a). Because each strand is coupled at the 5′ end to either the surface or the analyte, it is guaranteed that the analyte derivative is directed into the solution as it is schematically shown in Figure 1. (This would work the same way if both strands were coupled by the 3′ end.) An antibody against the analyte A was added, and its binding (Figure 1, 1b) is monitored by the sensor. Oligomer 2 is removed together with the bound antibody (Figure 1, 1c) by denaturation of the double strand. Oligomer 1 remains on the surface and can be loaded again, e.g., with Oligomer 3, an oligonucleotide conjugated at the 5′ end with a second hapten B (Figure 1, 2a). Again, binding of the corresponding antibody against B (Figure 1, 2b) can be performed, and consecutive denaturation of the hybrid (Figure 1, 2c) leaves the surface for new experiments. 756 BioTechniques

Specificity can be demonstrated by competition binding, and unspecific binding can be analyzed by running antibody against A on the B-modified surface and vice versa. In our example, we used 2,4-D as the first analyte and atrazin as the second. Both substances are herbicides, both inhibit the photosystem II of higher plants and are therefore of analytical interest in agriculture and environmental control. They are used here as model substances, because their chemical structures are completely different (Figure 1). 2,4-D was coupled to the DNA by its carboxylic group forming Oligomer 2. For atrazin, AHA was used as a derivative for conjugation (Oligomer 3). Figure 2A shows a real time measurement of hybridization of Oligomer 3 to the immobilized Oligomer 1 (Figure 2A, a) and the following addition of anti-atrazin-antibody (Figure 2A, b). The surface loading of binding sites (AHA) for the antibody can be calculated from the refractive index change caused by the oligonucleotide hybridization (2). The amount was 18 fmol/mm2, which is found to be nearly the maximum that can be achieved for oligonucleotides (2,23). The amount of antibody against atrazin added was

rather low (0.1 µg/mL = 0.7 nmol/L). Because of the short incubation time (Figure 2A, b to b′ = 4 min) no saturation of binding sites was achieved, the resulting surface coverage was only 0.7 fmol/mm2 of bound antibody. Regeneration of the surface was achieved by flushing 50 mM NaOH (Figure 2A, c). Figure 2B shows a series of measurements changing from one to another functionality according to the scheme given in Figure 1. In all cases, the complementary conjugated oligonucleotide

Figure 3. Example of an immunoassay for atrazin performed on an oligonucleotide-modified sensor chip as described in Figure 2 using Oligomer 3.

Figure 4. Examples of immunoassays for 2,4-D performed on an oligonucleotide-modified sensor chip as described in Figure 2 using Oligomer2. The surface loading with 2,4-D derivative was varied by changes in the amount of Oligomer2 during hybridization as indicated. For comparison, a competitive immunoassay with directly linked 2,4-D (without Oligo-Tag) is shown. Vol. 27, No. 4 (1999)

Short Technical Reports was added, followed by an incubation of the antibody in the presence and absence of the respective analyte. The doublestranded oligonucleotides were separated by a short flash of 50 mM NaOH after each incubation. The first two injections were made with Oligomer 2, the 2,4-D modified oligonucleotides, followed by specific antibodies against 2,4-D in the absence (Figure 2B, 1) and presence (Figure 2B, 2) of soluble 2,4-D. The same procedure was performed with Oligomer 3. The atrazin-specific antibody was added in high (1 µg/mL) and low (0.1 µg/mL) (Figure 2B, 3) concentration. In the presence of atrazin (0.1 µg/mL) (Figure 2B, 4), no binding was observed (Figure 2B, 5). After regeneration, the surface was free for other hybridization experiments. All experiments shown here were performed with the same concentration of oligonucleotides (1 µM). The experiments shown in Figure 2 were performed manually. Because

equilibrium is not reached within these experiments, the surface coverage varies because of varying incubation times. A more precise time regime achieved by complete automation leads to reproducible surface coverage with a standard deviation of 3%. Control experiments were made by incubation of anti-atrazine antibodies on a 2,4-Dmodified surface and vice versa. Unspecific binding has not been observed (data not shown). A competitive-binding assay performed in the same manner as described in Figure 2 is given in Figure 3. Without any optimization of the experimental conditions, the competitive assay for atrazin covers a range down to 1 ppb that might be compared to an optimized assay without any spacer (or tag) (5). In Figure 4, we present an assay against 2,4-D. The surface loading of 2,4-D derivatives is varied by changes in the concentration of Oligomer 2 (2,4-D-labeled). To obtain the same level of hybridization and to avoid any side effect from unpaired single strands, the concentration of oligomer was kept constant by addition of unlabeled Oligomer 4. The amount of Oligomer 2 was varied from 1–5 ng/mL during the hybridization period as indicated in Figure 4. The immunoassay shows similar performance as has been found without tag; comparison data are given in Figure 4 (open dots). The surface loading of the sensor does not influence the assay characteristic. However, it has an impact on the signal intensity, i.e., absolute surface coverage by antibodies. All measurements of this series have been done with one multiple regenerated surface. The method described makes use of the high stability of nucleic acids. DNA especially is designed by nature for information storage and is therefore a persistent molecule: the surface can be reloaded many times. In our experiments, we repeated the loading procedure more than 60 times. More stability can be expected from PNA even under a biologically active environment, since they are not degraded by enzymes (16). We have shown how to switch a functionalized surface, here a sensing device, from one functionalization to another, and here two different herbicides as haptens in an immunoassay.

The functionalities might be any hapten as well as any peptide or protein. Mixed surfaces may be created for multifunctional surfaces or for multitasking. Also, structured surfaces may be created, and the recently introduced DNA chips (10) might well be switched from an oligonucleotide array to an array of haptens or proteins, and their functionality might be changed arbitrarily. The stability of the surface can be regulated by the amount of matching bases. Thus, the structure can be varied by temperature or alternatively by means of more or less stringent washing conditions. This might be useful for the construction of multiparameter analytical tools that can be restricted more or less to parameters without change of hardware. This might also be useful for the construction of preparative tools, e.g., affinity chromatography that can be restricted consecutively to more specific binders. The density of functionalities may be varied in a broad range. The introduced method of anchoring functionalities might be called “Oligo-Tag”; its advantage over other tags is its high flexibility and potential of manipulation at several stages of an experiment. The length of the Oligo-Tag might be varied according to the application. If more stable tagging is desired, longer oligomers have to be used. However, above the limit of approximately 30 nucleotides (nt), discrimination of sequence deviations becomes less accurate. On the other hand, too short Oligo-Tags will be unreliable at least at elevated temperatures. As a rule of thumb, the Oligo-Tag length has to be chosen like the primer length in polymerase chain reaction (PCR) experiments. The usefulness of the Oligo-Tag is obvious for analytical tasks. Applications are not restricted to immunological or analytical problems but might be found in biotechnological surfacebound catalysis and synthesis. The potential application of the method might even be broadened by recent results of Barton et al. (11) on electron transfer along the helix axes of the double strand. That would make oligonucleotide tags the ideal linker for redox-active biomolecules on electrode surfaces. Oligo-Tags might also serve as linker for the recently developed aptamers (9) and ribozymes (12), since Vol. 27, No. 4 (1999)

Short Technical Reports the Oligo-Tag easily can be integrated in the production process of such nucleic acid-based molecular recognition elements. Experiments are under way to investigate these applications of the described Oligo-Tag. REFERENCES 1.Bickerstaff, G.F. 1996. Immobilization of Enzymes and Cells. Humana Press, Totowa. 2.Bier, F.F., F. Kleinjung and F.W. Scheller. 1997. Real time measurement of nucleic acid hybridization using evanescent wave sensors —steps towards the genosensor. Sens. Act. 38-39:78-82. 3.Bier, F.F., W. Stöcklein and R.D. Schmid. 1992. Direct observation of anti-atrazin antibody binding. GBF Monogr. 17:205-208. 4.Brandenburg, A., R. Polzius, F.F. Bier, U. Bilitewski and E. Wagner. 1996. Direct observation of affinity reactions by reflected mode operation of integrated optical grating coupler. Sens. Act. 30:55-59. 5.Brecht, A. and G. Gauglitz. 1995. Optical probes and transducers. Biosens. Bioelectron. 10:923-936. 6.Cantor, C.R. and P.R. Schimmel. 1980. Biophysical Chemistry Part III: The Behaviour of Biological Macromolecules, p. 1185-1239.

Freeman and Co., San Francisco. 7.Fránek, M., V. Kolár, M. Granátová and Z. Nevoránková. 1994. Monoclonal ELISA for 2,4-dichlorophenoxyacetic acid: characterization of antibodies and assay optimization. J. Agric. Food Chem. 42:1369-1374. 8.Giersch, T. and B. Hock. 1990. Production of monoclonal antibodies for the determination of s-triazines with enzyme immunoassays. Food Agric. Immunol. 2:85-97. 9.Gold, L., B. Polisky, O. Uhlenbeck and M. Yarus. 1995. Diversity of oligonucleotide functions. Annu. Rev. Biochem. 64:763-797. 10.Hoheisel, J.D. 1997. Oligomer-chip technology. Trends Biotechnol. 15:465-469. 11.Holmlin, R.E., P.J. Dandliker and J.K. Barton. 1997. Charge transfer through the DNA base stack. Angew. Chem. Int. Ed. Engl. 36:2714-2730. 12.Jones, J.T., S.-W. Lee and B.Y. Sullenger. 1996. Tagging ribozyme reaction sites to follow trans-splicing in mammalian cells. Nature Medicine 2:643-647. 13.Jönsson, U., L. Fägestam, B. Ivarsson, B. Johnsson, R. Karlsson, K. Lundh, S. Löfas, B. Persson et al. 1991. Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. BioTechniques 11:620-627. 14.Kleinjung, F., F.F. Bier, A. Warsinke and F.W. Scheller. 1997. Fibre-optic genosensor for specific determination of femtomolar DNA oligomers. Anal. Chim. Acta 350:51-59. 15.Mosbach, K. (Ed.). 1976. Immobilized enzymes. Methods Enzymol. 44:15-125. 16.Nielsen, P.E. 1995. Peptide nucleic acids. Annu. Rev. Biophys. Biomol. Struct. 24:167183. 17.Niemeyer, C.M., T. Sano, C.L. Smith and C.R. Cantor. 1994. Oligonucleotide-directed self-assembly of proteins: semisynthetic DNA-streptavidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates. Nucleic Acids Res. 22:55305539. 18.Peyman, A., E. Uhlmann, K. Wagner, S. Augustin, C. Weiser, D.W. Will and G. Breipohl. 1997. PHONA-PNA co-oligomers: nucleic acid mimetics with interesting properties. Angew. Chem. Int. Ed. Engl. 36:28092812. 19.Rosznyai, L.F., D.R. Benson, S.P.A. Fodor and P.G. Schultz. 1992. Photolithographic immobilisation of biopolymers on solid supports. Angew. Chem. Int. Ed. Engl. 34:799800. 20.Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. CSH Laboratory Press, Cold Spring Harbor, NY. 21.Scheller, F.W. and F. Schubert. 1992. Biosensors, p. 50-62. Elsevier Sci. Pub., Amsterdam. 22.Tiefenthaler, K. 1992. Integrated optical couplers as chemical waveguide sensors, p. 261289. In A.P.F. Turner (Ed.), Advances in Biosensors, Vol. 2. JAI Press, London. 23.Watts, H.J., D. Yeung and H. Parkes. 1995. Real-time detection and quantification of DNA hybridization by an optical biosensor. Anal. Chem. 67:4283-4289.

This work was supported by the Deutsche Forschungsgemeinschaft DFG (Sche 401/3-1) and the Federal Ministry of Education Science and Technology (Az. 0310862). The authors are grateful for the donation of monoclonal antibodies by Prof. B. Hock, Munich and Dr. M. Fránek, Brno, Czech Republic. Address correspondence to Frank F. Bier, Fraunhofer-Institute for Biomedical Engineering, Dept. Molecular Bioanalytics, Arthur-Scheunert-Allee 114116, D-14558 Bergholz-Rehbrücke, Germany. Internet: [email protected] Received 2 November 1998; accepted 7 June 1999.

Frank F. Bier, Frank Kleinjung, Eva Ehrentreich-Förster and Frieder W. Scheller University of Potsdam Potsdam, Germany

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