Investigations on the Specificity of DNA Aptamers Binding to

Apr 10, 2009 - targets so far (Mann, D.; Reinemann, C.; Stoltenburg, R.;. Strehlitz, B. Biochem. ... acids were tested to act as potential binding par...
0 downloads 0 Views 1MB Size
Download PDF
Anal. Chem. 2009, 81, 3973–3978

Investigations on the Specificity of DNA Aptamers Binding to Ethanolamine Christine Reinemann, Regina Stoltenburg, and Beate Strehlitz* UFZsHelmholtz Centre for Environmental Research, Permoserstrasse 15, D-04318 Leipzig, Germany In our previous work, we selected aptamers binding to ethanolamine, one of the smallest molecular aptamer targets so far (Mann, D.; Reinemann, C.; Stoltenburg, R.; Strehlitz, B. Biochem. Biophys. Res. Commun. 2005, 338, 1928-1934). Two representatives of these aptamers (EA#14.3 and EA#9.4) were analyzed regarding their specificity. Ethanolamine is a very small organic molecule (Mw ) 61.08) with biological, medical, and industrial relevance. Its small size represented a challenge for aptamer development, as ethanolamine only consists of a short carbon chain (2C) and two functional groups (amino and hydroxyl group). Related organic molecules, ethanolamine derivatives, and some amino acids were tested to act as potential binding partners for these aptamers. In this way we were able to determine the exact binding domain within the target. The results revealed that both aptamers bind to various molecules, which contain a freely accessible ethyl- or methylamine group. In contrast to the amino group (in a primary, secondary, or tertiary amine) the hydroxyl group was not necessary for the aptamer binding. The aptamers were not able to bind to negatively charged organic molecules, despite containing an ethyl- or methylamine group, nor did they bind to molecules with quaternary amines. The selected ethanolamine binding aptamers are useful for the detection of molecules containing accessible ethyl- or methylamine groups; they can be used as linker elements to immobilize a target molecule of interest on a surface or to purify targets from complex samples. With the development of the SELEX process (systematic evolution of ligands by exponential enrichment) in 1990, a powerful technique for screening very large nucleic acid libraries has been provided in order to select highly specific oligonucleotide ligands with high affinity to several targets.1,2 These ligands, also referred to as aptamers, form a specific three-dimensional structure based on their primary sequence; they recognize, and bind to their target by molecular shape complementarities, stacking of aromatic rings, electrostatic, or van der Waals interactions, and hydrogen bondings.3 Since 1990, aptamers were selected for different classes of targets from small organic molecules, peptides, proteins, carbo* To whom correspondence should be addressed. Fax: +49 341 235451764. E-mail: [email protected]. (1) Tuerk, C.; Gold, L. Science 1990, 249, 505–510. (2) Ellington, A. D.; Szostak, J. W. Nature (London) 1990, 346, 818–822. (3) Hermann, T.; Patel, D. J. Science 2000, 287, 820–825. 10.1021/ac900305y CCC: $40.75  2009 American Chemical Society Published on Web 04/10/2009

hydrates, antibiotics to complex target structures like whole cells or viruses.4-9 The possibility to obtain aptamers for a wide variety of targets has contributed to a wide range of application of aptamers. The research on medical applications of aptamers as diagnostic and therapeutic agents has increased in recent years.10-13 Moreover, aptamers are attractive as affinity probes for diverse analytical applications, such as those based on chromatography, mass spectrometry, flow cytometry, image analysis, detection assays, and biosensors.14,15 Aptamers can also be used as molecular recognition elements for different substances in a wide area of environmental analysis. In this regard, the selection of aptamers for small organic molecules is of special interest. Several different aptamers, which were selected for small targets like, e.g., organic or fluorescent dyes, nucleotides, nucleobases, cofactors, amino acids, neurotransmitter, or natural products, have been described in literature.4,5,16,17 In our previous work, we selected aptamers binding to ethanolamine.18 Ethanolamine is a very small organic molecule (Mw ) 61.08) with biological, medical, and industrial relevance. It is involved in the biosynthesis of acetylcholine, known as component in phospholipids of biomembranes and in lipopolysaccharides of Gram-negative bacteria, and is associated with the storage disease ethanolaminosis. Moreover, it is used for scrubbing certain acidic gases and in surface-active agents, (4) Klussmann, S. The Aptamer Handbook. Functional Oligonucleotides and Their Applications; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2006. (5) Famulok, M. Curr. Opin. Struct. Biol. 1999, 9, 324–329. (6) Daniels, D. A.; Chen, H.; Hicke, B. J.; Swiderek, K. M.; Gold, L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 15416–15421. (7) Blank, M.; Weinschenk, T.; Priemer, M.; Schluesener, H. J. Biol. Chem. 2001, 276, 16464–16468. (8) Pan, W.; Craven, R. C.; Qiu, Q.; Wilson, C. B.; Wills, J. W.; Golovine, S.; Wang, J. F. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 11509–11513. (9) Hamula, C. L. A.; Zhang, H.; Guan, L. L.; Li, X. F.; Le, X. C. Anal. Chem. 2008, 80, 7812–7819. (10) Proske, D.; Blank, M.; Buhmann, R.; Resch, A. Appl. Microbiol. Biotechnol. 2005, 69, 367–374. (11) Lee, J. F.; Stovall, G. M.; Ellington, A. D. Curr. Opin. Chem. Biol. 2006, 10, 282–289. (12) Brody, E. N.; Gold, L. J. Biotechnol. 2000, 74, 5–13. (13) Ng, E. W. M.; Shima, D. T.; Calias, P.; Cunningham, E. T.; Guyer, D. R.; Adamis, A. P. Nat. Rev. Drug Discovery 2006, 5, 123–132. (14) Tombelli, S.; Minunni, A.; Mascini, A. Biosens. Bioelectron. 2005, 20, 2424– 2434. (15) Hamula, C. L. A.; Guthrie, J. W.; Zhang, H. Q.; Li, X. F.; Le, X. C. TrAC, Trends Anal. Chem. 2006, 25, 681–691. (16) Bruno, J. G.; Carrillo, M. P.; Cadieux, C. L.; Lenz, D. E.; Cerasoli, D. M.; Phillips, T. J. Mol. Recognit. 2009, 22, 197-204. (17) Bruno, J. G.; Carrillo, M. P.; Phillips, T.; King, B. In Vitro Cell. Dev. Biol. Anim. 2008, 44, 63–72. (18) Mann, D.; Reinemann, C.; Stoltenburg, R.; Strehlitz, B. Biochem. Biophys. Res. Commun. 2005, 338, 1928–1934.

Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

3973

Figure 1. Sequences and secondary structures: the sequences of the analyzed ethanolamine binding aptamers EA#14.3 and EA#9.4. The consensus sequence is marked in bold letters. The energetic favorable secondary structures of aptamer EA#14.3 and EA#9.4, determined by the Internet tool “mfold” (ref 19) are shown. Primer regions (nucleotides 1-18 and 79-96) are highlighted in gray, and the consensus sequence is marked by circles.

cleaners, emulsifiers, or cosmetic products. Ethanolamine is one of the smallest molecular aptamer targets so far. We selected ethanolamine binding DNA aptamers from an initial random oligonucleotide library (96-mers) by the FluMagSELEX process.18 Ethanolamine was immobilized on magnetic beads for its use as target in the aptamer selection process. Thirty-seven aptamer clones from the selected pool were analyzed. There were two main groups, I (e.g., EA#14.3) and II (e.g., EA#9.4), with 21 and 11 homologous aptamer sequences, respectively (Figure 1).19 A third group (III) was represented only by one aptamer sequence. In all three groups, a G-rich consensus sequence was found, which could form a typical G-quartet structure. Binding studies with ethanolaminecoated magnetic beads as target revealed high affinities of the selected aptamers to their target with dissociation constants in the low nanomolar range (6-10 nmol L-1). In the present work, we characterize the selected ethanolamine binding aptamers with focus on their specificity. Therefore, we tested the binding activities of the aptamers to several structurally and functionally similar molecules and to some amino acids. EXPERIMENTAL SECTION Materials and Instruments. Fluorescein-labeled ethanolamine aptamers EA#14.3 and EA#9.4 were prepared by polymerase chain reaction (PCR) followed by denaturing polyacrylamide gel electrophoresis (PAGE) and gel elution as described in detail in Stoltenburg et al.20 Ethanolamine, di- and triethanolamine, 4-amino-1-hexanol, 4-amino-1-butanol, phenylmethylamine, methylamine, ethylamine, phenylethylamine phosphocolamine, cho(19) Zuker, M. Nucleic Acids Res. 2003, 31, 3406–3415. (20) Stoltenburg, R.; Reinemann, C.; Strehlitz, B. Anal. Bioanal. Chem. 2005, 383, 83–91.

3974

Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

line chloride, serine, asparagine, and histidine were purchased from Fluka (Germany). Methylaminoethanol, phenoxyethylamine, CDP-ethanolamine, β-alanine, arginine, and lysine were obtained from Sigma (Germany). Glycine, glutamine, ethanol, di- and triethylamine were purchased from Merck (Germany). M-280 tosyl-activated Dynabeads (magnetic beads; Invitrogen/Dynal, U.K.) were used for immobilization of ethanolamine. The fluorescence detection for quantification of the fluorescein-labeled aptamers was performed in 96-well black microtiter plates (NUNC, Germany) using the Wallac multilabel counter 1420 Victor2V (Perkin-Elmer, Germany) under the following conditions: prompt fluorometry, excitation filter 485 nm, emission filter 535 nm, time 1 s, CW lamp energy 15 000. The sample volume was 100 µL/well. Calibration curves were prepared with fluoresceinlabeled ssDNA of the random oligonucleotide library (96-mers). Methods. Affinity Elution Assays. The ability of the ethanolamine aptamers to bind to free ethanolamine in solution and to related organic molecules was tested by affinity elution assays. Ethanolamine was immobilized on tosyl-activated magnetic beads according to manufacturer’s instructions. Amounts of 1-2 × 107 of these modified beads were used in each affinity elution assay. At first, 10-15 pmol of fluorescein-labeled aptamers EA#14.3 or EA#9.4 were incubated with washed ethanolamine-coated magnetic beads in 500 µL of binding buffer (100 mmol L-1 NaCl, 20 mmol L-1 Tris-HCl pH 7.6, 2 mmol L-1 MgCl2, 5 mmol L-1 KCl, 1 mmol L-1 CaCl2, 0.02% Tween 20) for 30 min at 21 °C while shaking. Before this binding step the aptamers were denatured at 90 °C for 8-10 min, cooled down by incubation on ice for 15 min and at room temperature (RT) for 5 min. After the binding reaction, the unbound aptamers were removed from the aptamer-bead complexes by several washing steps with binding buffer. Thereafter, the aptamer-bead

complexes were incubated for 15 min at 21 °C with shaking in 500 µL of binding buffer containing 10 mmol L-1 ethanolamine or related organic molecules to be investigated. During this step some of the bead-bound aptamers are released from the beads according to the affinity of the aptamers to the respective molecules in solution. The supernatant represents the affinity elution fraction. The beads were washed three times with 200 µL of binding buffer (washing fraction). After that, all of the remaining aptamers were completely eluted from the beads by twice-repeated heat treatment at 80 °C for 7 min in 100 µL of binding buffer (heat elution fraction). The amounts of aptamers in each elution fraction (affinity elution, washing steps, and heat elution) were determined by fluorescence measurement and calculated using a calibration curve. RESULTS AND DISCUSSION Specificity of the Aptamers EA#14.3 and EA#9.4. The ethanolamine binding aptamers EA#14.3 and EA#9.4 are representatives of the two main sequence groups (I and II) identified from the selected aptamer pool after the SELEX process described in our previous work.18 Both aptamers are able to bind very tightly (KD’s of 6 and 10 nmol L-1)18 to their target ethanolamine which was immobilized on magnetic beads. Ethanolamine is a very small and simple aptamer target characterized by a short carbon chain (2C) and two functional groups (amino group, hydroxyl group). Affinity elution assays were used to determine the binding ability of aptamers EA#14.3 and EA#9.4 to free ethanolamine in solution, to ethanolamine derivatives, and to related organic molecules. In these assays the binding complexes consisting of ethanolamine-coated magnetic beads and bound aptamers were incubated in test solutions containing an excess of ethanolamine or another potential target molecule. The organic molecules in the test solutions compete against ethanolamine-coated magnetic beads for binding to the aptamers. Only if cross-reactivity exists, the bead-bound aptamers are released from their binding to ethanolamine on the magnetic beads and undergo the binding to the organic molecules in the test solution, representing the affinity elution fraction. For determining the total amount of eluted aptamers, the amount of aptamers present in the affinity elution fraction has to be added to the amounts contained in the washing and heat elution fractions, cf. the Methods section. The results of the affinity elution assays are displayed in the following figures (Figures 2 and 3) in which the amounts of aptamers in the affinity elution fractions are shown as percentages of the total amount of aptamers eluted from the beads. Figure 2 shows the results of structurally related organic molecules to the selection target ethanolamine, whereas in Figure 3 the values of functionally related molecules and selected amino acids are displayed. Both aptamers EA#14.3 and EA#9.4 were able to bind to ethanolamine in solution as free, nonimmobilized target. During the affinity elution step, 84% of aptamer EA#14.3 and 79% of aptamer EA#9.4 in complexes with the beads were eluted (Figure 2). Binding buffer without any additional organic molecules was used as control. Only 13-20% of the bead-bound aptamers in the binding complex were nonspecifically washed away by the buffer solution during the affinity elution step. Among the tested ethanolamine derivatives, the best binding ability of both aptamers was found to diethanolamine. Amounts of 95-96% of bead-bound

aptamers were eluted by diethanolamine during the affinity elution step. In contrast, only 55-64% of the bead-bound aptamers could be eluted by triethanolamine. Ethanolamine-related organic molecules with an extension of the carbon chain like 4-amino-1-butanol and 4-amino-1-hexanol are very good targets for the aptamers EA#14.3 and EA#9.4 (97-99% eluted aptamers from the beads; Figure 2). With regard to the functional groups present in ethanolamine (amino group, hydroxyl group) organic molecules with varying structures, partially containing only one of these groups, were used as potential binding partners for the aptamers in the affinity elution assays (Figure 2). Both aptamers EA#14.3 and EA#9.4 were not able to bind to ethanol (no amino group). Very good binding of the aptamers was observed to diethylamine and triethylamine (no hydroxyl group) and also to phenoxyethylamine and methylaminoethanol. Amounts of 87-99% of beadbound aptamers were eluted by these organic molecules during the affinity elution step. Ethylamine, methylamine, phenylethylamine, and phenylmethylamine have also proved to be aptamer binding partners but could not elute such high amounts of beadbound aptamers (54 - 75% elution of aptamer EA#14.3, 30- 46% elution of aptamer EA#9.4). Figure 3 shows the results of the affinity elution assays using the following, biologically relevant substances as potential target molecules: choline (chloride), serine, CDP-ethanolamine, and phosphocolamine. The last two substances are involved in the biosynthesis of phospholipids (phosphatidyl ethanolamine). Choline and serine are, like ethanolamine, head groups for phospholipids. The aptamers EA#14.3 and EA#9.4 were not able to bind to these substances. The small amounts of aptamers in the elution fractions (7-23%) are caused by unspecific washing effects. Beside serine, further amino acids like glycine, β-alanine, lysine, arginine, asparagine, glutamine, and histidine were tested in the affinity elution assays (Figure 2). The aptamers EA#14.3 and EA#9.4 could only bind to lysine (76-94% eluted aptamers) and arginine (38-57% eluted aptamers), both characterized by a side chain with an amino group. None of the other amino acids could function as target for these aptamers. Discussion. The present work focused on the investigation of the specificity of the ethanolamine binding aptamers EA#14.3 and EA#9.4. Affinity elution assays were used for a simple and fast screening of several organic molecules to act as potential targets for the aptamers. Both aptamers showed comparable binding characteristics with the tested substances. The specificity profile is almost identical. For a better comparison of the experiments, the amounts of affinity eluted aptamers are given as percentage of the total amount of all eluted aptamers per assay. Generally, this percentage for aptamer EA#9.4 is less than for aptamer EA#14.3., because aptamer EA#9.4 is able to bind in higher rates to ethanolamine-coated magnetic beads than aptamer EA#14.3. (see Mann et al.18). Very good binding abilities of both aptamers were observed to organic molecules similar to ethanolamine but with an extension of the carbon chain. EA#14.3 and EA#9.4 were also able to bind to the ethanolamine derivatives diethanolamine as a secondary amine and triethanolamine as a tertiary amine. The best results were obtained for diethanolamine, which is structured like the immobilized form of ethanolamine (generating a secondary amine) used in the aptamer selection process. The slightly poorer binding Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

3975

Figure 2. Affinity elution assays: affinity elution assays using aptamers EA#14.3 and EA#9.4. The ability of the EA aptamers to bind to free ethanolamine in solution and to related organic molecules was tested by affinity elution assays. For each assay, the aptamers labeled with fluorescein were first bound to ethanolamine-coated magnetic beads. The washed aptamer-bead complexes were then incubated in a test solution containing an excess (10 mmol L-1) of ethanolamine or related organic molecules in binding buffer (BB). Some of the bead-bound aptamers are released from the beads into the supernatant and bind due to cross-reactivity to the substance in the test solution (affinity elution step). Afterward the beads were washed, and the remaining aptamers were completely eluted from the beads by heat treatment. The amounts of aptamers in each elution fraction were determined by fluorescence measurement and calculated using a calibration curve. The results of the elution fractions are shown as percentages of the total amount of aptamers eluted from the ethanolamine-coated magnetic beads. The specific structural features of the substances used in the test solutions during the affinity elution steps are shown above the diagrams.

to triethanolamine is possibly caused by steric hindrance. The affinity elution assays have further shown that, in contrast to the hydroxyl group, which is not necessary for the aptamer binding, 3976

Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

the amino group coupled to a short carbon chain in an organic molecule is essential for binding of the aptamers EA#14.3 and EA#9.4. Thus, the aptamers showed binding abilities to organic

Figure 3. Affinity elution assays: affinity elution assays using aptamers EA#14.3 and EA#9.4. The ability of the EA aptamers to bind to free ethanolamine in solution and to related organic molecules was tested by affinity elution assays. For each assay, the aptamers labeled with fluorescein were first bound to ethanolamine-coated magnetic beads. The washed aptamer-bead complexes were then incubated in a test solution containing an excess (10 mmol L-1) of ethanolamine or related organic molecules in binding buffer (BB). Some of the bead-bound aptamers are released from the beads into the supernatant and bind due to cross-reactivity to the substance in the test solution (affinity elution step). Afterward the beads were washed, and the remaining aptamers were completely eluted from the beads by heat treatment. The amounts of aptamers in each elution fraction were determined by fluorescence measurement and calculated using a calibration curve. The results of the elution fractions are shown as percentages of the total amount of aptamers eluted from the ethanolamine-coated magnetic beads. The specific structural features of the substances used in the test solutions during the affinity elution steps are shown above the diagrams.

molecules lacking the hydroxyl group (e.g., methylamine, ethylamine, di- and triethylamine), whereas no binding was found to ethanol which contains no amino group. These results indicate

that the aptamers EA#14.3 and EA#9.4 are specific for the ethylor methylamine group. Even if there are ring structures close to the binding region of the target (ethyl- or methylamine group), Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

3977

aptamers were able to bind to the targets, even though with smaller rates (see results of phenylethylamine and phenylmethylamine). Higher amounts of eluted aptamers were obtained with phenoxylethylamine which additionally contains an oxygen atom between the target binding region and the phenyl ring structure. The affinity elution assays identified some negative influences on the binding of the aptamers under investigation to potential targets. Both aptamers were not able to bind to organic molecules which are negatively charged due to phosphate groups in addition to containing an ethylamine group (CDP-ethanolamine, phosphocolamine). This corresponds to results by other authors stating poor binding properties of aptamers to phosphate groups.21 Nevertheless, there are examples of successful aptamer bindings to molecules containing phosphate groups, like, e.g., nucleotides,22-24 thiamine pyrophosphate,25 or methylphosphonic acid.16 In some cases these aptamers bind particularly to the phosphate moieties.24-26 No binding was also found to choline (chloride) as a quaternary ammonium salt. In general, the binding of aptamers to their targets occur by a combination of molecular shape complementarities, stacking interactions, electrostatic interactions, and especially by specific hydrogen bondings.3 In the case of choline (chloride), a hydrogen bonding between the quaternary saturated amino group and the aptamers investigated here is not possible. Several amino acids were also tested in the affinity elution assays. As amino acids have both an amino as well as a carboxyl group, they are known as zwitterions in polar solutions. Amino acids differ in their respective side chains. The aptamers were only able to bind to lysine and arginine containing an additional amino group in the side chain. The results of the affinity elution assays with histidine as potential target suggest furthermore that the aptamers supposedly cannot bind to heterocyclic compounds containing carbon and nitrogen atoms within a ring structure (like the imidazole ring of histidine). Test results with glutamine and asparagine indicate that carboxamide groups are also unsuited for binding by the selected aptamers. To summarize the results of the affinity elution assays, the aptamers EA#14.3 and EA#9.4 are specific for ethyl- or methy(21) Saran, D.; Frank, J.; Burke, D. H. BMC Evol. Biol. 2003, 3, 26. (22) Huizenga, D. E.; Szostak, J. W. Biochemistry 1995, 34, 656–665. (23) Davis, J. H.; Szostak, J. W. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11616– 11621. (24) Sazani, P. L.; Larralde, R.; Szostak, J. W. J. Am. Chem. Soc. 2004, 126, 8370–8371. (25) Noeske, J.; Richter, C.; Stirnal, E.; Schwalbe, H.; Wohnert, J. ChemBioChem 2006, 7, 1451–1456. (26) Bruno, J. G.; Carrillo, M. P.; Phillips, T.; Vail, N. K.; Hanson, D. J. Fluoresc. 2008, 18, 867–876.

3978

Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

lamine group. Therefore, the aptamers are not only able to bind to ethanolamine but also to larger organic molecules which contain at least a methylamine group. Structurally more complex molecules like peptides or proteins were not tested in this work as potential binding partners. But it is supposed that the aptamers also cross-react with those complex molecules if an ethyl- or methylamine group is freely accessible for binding. Accordingly, the selected ethanolamine binding aptamers offer a high specificity for the mentioned functional groups, but wider substance specificity. Due to these characteristics possible applications arise. Molecules containing accessible ethyl- or methylamine groups can be detected by these aptamers. However, because of this group specificity the aptamers are not able to distinguish between different molecules containing one of these groups and occurring in parallel in the test solution, e.g., in a real sample. That means that these aptamers have lower substance specificity because of the high group specificity. Furthermore, the aptamers could be used as linker elements to immobilize target molecules of interest on a surface or to purify targets bearing the mentioned groups. Thus, they could be helpful for prepurification steps of complex samples. The aptamers are used already routinely in our laboratory to prove the occupancy of immobilization matrixes whose nonoccupied binding sites are blocked with ethanolamine. The proof occurs by indirect measurements, and the rate of matrix-bound EA aptamers displays the rate of nonoccupied binding sites. The results respecting the high group specificity and connected low substance specificity of these aptamers are also important for further SELEX experiments. Because of the simple structure of the ethyl- or methylamine group, their presence in a multitude of larger or more complex organic molecules is expected. Therefore, it is possible to coselect the described ethanolamine binding aptamers repeatedly in other SELEX processes with dependence on the structural features of the targets. Negative selection steps should be introduced to minimize such a coselection and to direct the aptamer selection to other potential binding regions in the target molecules. ACKNOWLEDGMENT We thank Andre´ Schuster, Ines Kahnt, and Christina Petzold for their excellent assistance. We also thank Nadia Nikolaus for critical reading of the manuscript. The first two authors contributed equally to this work. Received for review February 9, 2009. Accepted March 24, 2009. AC900305Y