AC Research
Accelerated Articles Anal. Chem. 1996, 68, 4309-4311
Patterned Staining by Fluorescein-Labeled Oligonucleotides Obtained by in Vitro Selection Naoki Kawazoe, Yoshihiro Ito,* and Yukio Imanishi
Department of Material Chemistry, Faculty of Engineering, Kyoto University, Kyoto, 606-01, Japan
We describe a new means of surface staining using a fluorescent oligonucleotide. A folic acid-specific, fluorescent-labeled oligonucleotide was selected from a group of randomly sequenced oligonucleotides by repeated elution through a folic acid-immobilized column and amplified by the polymerase chain reaction. We then examined the oligonucleotide scattered on a polyester film with folic acid immobilized in a defined pattern, by means of laser fluorescence microscopy. The results showed that the selected oligonucleotide fluoresced exactly following the pattern of immobilized folic acid.
Selective binding through molecular recognition is the basis of many chemical analyses and separations, including those using enzymes, antibodies, or selective chelators. Recently, systematic evolution of ligands by exponential enrichment (SELEX) or in vitro selection was devised for the identification of high-affinity oligonucleotide ligands to target molecules.1-3 Nucleic acids are capable of adopting intrinsic three-dimensional structures. A large library of random sequence single-stranded oligonucleotides, whether DNA or RNA, is therefore likely to populate a significant fraction of shape space. Oligonucleotide ligands that have high affinity for a target molecule can be isolated by iterative rounds of affinity selection and amplification from a large randomized sequence set. A close analogy to the oligonucleotide ligand is the antibody.4 Antibodies are proteins that develop molecular recognition by in vivo exposure of the unspecified immunoglobulin to a target through a naturally or artificially induced immunogenic response. The selection of an oligonucleotide ligand is analogous to the immunogenic process. (1) (2) (3) (4)
Robertson, D. L.; Joyce, G. F. Nature 1990, 344, 467-468. Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818-822. Tuerk, C.; Gold, L. Science 1990, 219, 505-510. McGown, L.; Joseph, M. J.; Pitner, J. B.; Vonk, G. P.; Linn, C. P. Anal. Chem. 1995, 67, 663A-668A.
S0003-2700(96)00684-1 CCC: $12.00
© 1996 American Chemical Society
Oligonucleotide ligands offer several potential advantages over traditional antibody-based reagents. They are not derived from living organisms and can be rapidly, reproducibly, and accurately synthesized by automated processes. The covalent attachment of dyes to oligonucleotide ligands is relatively simple and may be highly specific when the dyes are placed at one or more locations on the ligand. The present study demonstrated the usefulness of in vitro-selected oligonucleotide for patterned staining. There is increasing scientific and commercial interest in the development of techniques to selectively immobilize macromolecules at specific locations on solid supports.5 The potentialapplications for such products range from microbiosensors to cell guidance for artificial organ development.6-14 However, oligonucleotide ligands have not been immobilized, although the ligands have great potential. This study showed that oligonucleotide ligands to folic acid were selected in vitro and that a defined pattern of immobilized folic acid was precisely reproduced by the fluorescein-labeled ligands. (5) Sundberg, S. A.; Barrett, R. W.; Pirrung, M.; Lu, A. L.; Kiangsoontra, B.; Holmes, C. P. J. Am. Chem. Soc. 1995, 117, 12050-12057. (6) Britland, S.; Perez-Arnaud, E.; Clark, P.; McGinn, B.; Connolly, P.; Moores, G. Biotechnol. Prog. 1992, 8, 155-160. (7) Stegner, D. A.; Georger, J. H.; Dulcey, C. S.; Hickmann, J. J.; Rudolph, A. S.; Nielsen, J. B.; McCort, S. M.; Calvert, J. M. J. Am. Chem. Soc. 1992, 114, 8435-8442. (8) Lopez, G. P.; Albers, M. W.; Schreiber, S. L.; Carroll, R.; Peralta, E.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 5877-5878. (9) Spargo, B. J.; Testoff, M. A.; Nielsen, T. B.; Hickmann, J. J.; Rudolph, A. S. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11070-11074. (10) Singhvi, R.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I. C.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696-698. (11) Rahn, J. R.; Hallock, R. B. Langmuir 1995, 11, 650-654. (12) Bekos, E. J.; Ranieri, J. P.; Aebischer, P.; Gardella, J. A., Jr.; Bright, F. V. Langmuir 1995, 11, 984-989. (13) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Angew. Chem., Int. Ed. Engl. 1995, 34, 91-93. (14) Sangodkar, H.; Sukeerthi, S.; Srinivasa, R. S.; Lal, R.; Contactor, A. Q. Anal. Chem. 1996, 68, 779-783.
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Figure 1. Schematic illustration of the in vitro selection cycle. DNA random oligomers (top) consist of 60 random bases flanked by defined regions. Asymmetric PCR-amplified DNA oligomers with various shapes are selected by affinity chromatography. Bound DNA is eluted and amplified by PCR, and then the cycle is repeated.
EXPERIMENTAL SECTION Materials. Folic acid was purchased from Nacalai Tesque Ltd. (Kyoto, Japan). Agarose gel derivatized with amino groups (EAH Sepharose 4B) and deoxynucleoside triphosphates (dNTPs) were purchased from Pharmacia Biotech (Upsala, Sweden). Nonlabeled and fluorescein-labeled oligonucleotides were custom synthesized by means of solid phase chemistry by Sawaday Technology (Tokyo, Japan). A thermoresistant DNA polymerase (Taq polymerase) was purchased from Perkin-Elmer (Amplitaq). Poly(allylamine) was purchased from Nittobo (Tokyo, Japan). In Vitro Selection of Oligonucleotides to Folic Acid. The in vitro selection proceeded essentially as described.15 The procedure is schematically shown in Figure 1. Folic acid was immobilized on the agarose gel at a concentration of 0.6 µmol/mL of gel. For the starting selection on folic acid, a 104-mer oligonucleotide with a random insert of 60 nucleotides, 5′-TAGGGAATTCGACGGATCC-N60-CTGCAGGTCGACGCATGCGCC-3′, was amplified using the primers 5′TAATACGACTCAACTATAGGGAATTCGTCGACGGAT-3′ (P1) and 3′-GTCCAGCTGCGTACGCGCC-5′ (P2). Oligonucleotide (5 µg) was amplified by means of 30 polymerase chain reaction (PCR) cycles (one cycle: 94 °C, 30 s; 55 °C, 30 s; 72 °C, 30 s) in 100 µL of PCR mix [10 mM Tris-HCl, pH ) 8.3; 50 mM KCl; 1.5 mM MgCl2; 0.001% gelatin; 0.02 mM dNTPs; primers P1 (0.5 µM) and P2 (0.5 µM)]. The amplified double-strand oligonucleotide was extracted with phenol/chloroform/diethyl ether and then precipitated with ethanol. The primers were removed by ultrafiltration. Subsequently, a single-strand oligonucleotide was (15) Ellington, A. D.; Szostak, J. W. Nature 1992, 355, 850-852.
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obtained by 45 PCR cycles (one cycle: 94 °C, 30 s; 55 °C, 30 s; 72 °C, 30 s) of the double-strand oligonucleotide using the P1 primer.16 The PCR single-strand oligonucleotide was extracted with phenol/chloroform/diethyl ether and precipitated with ethanol. A 500 µL sample of the single-strand oligonucleotide pool (6 µg) in binding buffer (0.5 M LiCl; 10 mM Tris-HCl, pH ) 7.6; 1 mM MgCl2) was loaded onto the folic acid column, which was then rinsed with 5 mL of binding buffer (five column volumes). Aptamers were eluted with three column volumes of 3 mM folic acid in binding buffer. The eluted single-strand oligonucleotide was precipitated with ethanol in the presence of 100 µg of glycogen, washed with 70% ethanol, and dissolved in 40 µL of water. The single-strand oligonucleotide was amplified by PCR and used as the input for the next selection. These processes were repeated several times. Fluorescein Labeling of the Selected Oligonucleotide. The selected single-strand oligonucleotide was labeled with fluorescein by means of PCR amplification as described.17 The oligonucleotide (5 µg) was amplified by 30 cycles of PCR (one cycle: 94 °C, 30 s; 55 °C, 30 s; 72 °C, 30 s) in 100 µL of PCR mix [10 mM Tris-HCl, pH ) 8.3; 50 mM KCl; 1.5 mM MgCl2; 0.001% gelatin; 0.02 mM dNTPs; 5′-fluorescein-labeled P1 primer (0.5 µM) instead of P1 primer]. Patterned Immobilization of Folic Acid. Folic acid was immobilized on a poly(ethylene terephthalate) film in which poly(allylamine) was already immobilized over a specific area as described by Sugawara and Matsuda.18-20 The procedure is shown in Figure 2. Poly(allylamine) containing phenyl azide groups was synthesized by mixing polyallyamine and azidobenzoic acid in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (water-soluble carbodiimide, WSC). The product was purified by ultrafiltration. The polymer was dissolved in water (0.5 mg/mL) and spread over a poly(ethylene terephthalate) film (5 mm × 2 mm). After drying in air for 3 h, the film was irradiated in the presence of a photomask with ultraviolet light for 15 s and washed with water for 24 h. Folic acid was then bound to the poly(allylamine) derivatives attached to the surface. The film was immersed in 1 mL of folic acid (3 mg/mL) in the presence of WSC (25 mg) for 2 h at room temperature and washed with water until release of folic acid was undetectable by ultraviolet absorption. Staining and Observation of Patterned Surface. The film with immobilized folic acid was immersed in a solution of the labeled oligonucleotide (100 ng/mL) for 20 min at room temperature. Thereafter, the film was washed with water several times. The film was observed using a laser fluorescence microscope (BioRad, Hercules, CA). RESULTS AND DISCUSSION The amount of oligonucleotides eluted from the column packed with folic acid bound to a gel was monitored by means of ultraviolet absorption. The oligonucleotides with random sequences scarcely adsorbed on the gel. However, 1% of the oligonucleotide molecules bound to the gel after two selections, (16) Higuchi, R.; von Beroldingen, C. H.; Sensabaugh, C. F.; Erlich, H. A. Nature 1988, 332, 543-546. (17) Smith, L. M.; Fung, S.; Hunkapiller, M. W.; Hunkapiller, T. J.; Hood, L. E. Nucleic Acids Res. 1985, 13, 2399-2412. (18) Sugawara, T.; Matsuda, T. Macromolecules 1994, 27, 7809-7814. (19) Matsuda, T.; Sugawara, T. Langmuir 1995, 11, 2272-2276. (20) Matsuda, T.; Sugawara, T. Langmuir 1995, 11, 2267-2271.
Figure 3. Patterned staining of folic acid by fluorescein-labeled oligonucleotides. Oligonucleotides selected were labeled with fluorescein and adsorbed onto regions covered with immobilized folic acid.
Figure 2. Schematic illustration of the patterned immobilization of folic acid. A film was coated with poly(allylamine) carrying phenyl azide groups. The film was photoirradiated in the presence of a photomask. Noncovalently adsorbed poly(allylamine) was removed by washing. Folic acid was coupled to the immobilized poly(allylamine) by water-soluble carbodiimide.
7% after three selections, and about 30% after four selections. There was a significant improvement in the ability of the oligonucleotides to bind to immobilized folic acid as selection proceeded. The selected oligonucleotides were labeled with fluorescein by means of PCR using the fluorescent-labeled primer. Oligonucleotides with random sequences were also labeled with fluorescence as a control. Figure 3 shows the surface stained by the labeled DNAs. The labeled oligonucleotides adsorbed onto the folic acid-immobilized surface, whereas the labeled control oligonucleotides did not. This result indicated that the selected oligonucleotides specifically recognized folic acid. In this investigation, we stained the folic acid-immobilized region by noncloned oligonucleotide ligands that had various
different three-dimensional structures and bound to folic acid at different binding sites. The antibodies used in immunostaining or immunoassays can be also categorized into two kinds: one is polyclonal, composed of a heterogeneous mixture of immunoglobulins with binding affinities for several determinant structures on the target molecule, and the other is monoclonal, which is a homogeneous, pure species of immunoglobulin with selected specificity for a unique determinant on the target. Although the homogeneity of monoclonal antibodies is advantageous because of their reproducibility and predictability, polyclonal antibodies are frequently more effective in immunoassays. Our oligonucleotide ligands were not cloned. Heterogeneous sensor molecules are superior for this type of staining, because of their multirecognition sites. Procedures involving the molecular recognition of oligonucleotide ligands will replace the traditional techniques using antibodies. ACKNOWLEDGMENT We thank Drs. T. Matsuda and S. Nakayama of the National Cardiovascular Center Research Institute for their valuable suggestions on the patterned immobilization and Dr. M. Ueda of the Department of Synthetic Chemistry and Biological Chemistry of Kyoto University for his helpful discussions.
Received for review July 15, 1996. Accepted October 15, 1996.X AC960684M
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Abstract published in Advance ACS Abstracts, November 15, 1996.
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