Anal. Chem. 1998, 70, 3510-3512
Technical Notes
Competitive Binding Assay for Thyroxine Using in Vitro Selected Oligonucleotides Yoshihiro Ito,*,†,‡ Satoshi Fujita,§ Naoki Kawazoe,§ and Yukio Imanishi†
Graduate School of Materials Science, NAIST, Ikoma, 630-0101 Japan, PRESTO, JST, Hikaridai 1-7, Seika-cho, Kyoto, 619-0237 Japan, and Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 606-8501 Japan
A new binding assay that uses oligodeoxyribonucleotides (DNAs) obtained by the in vitro selection method, instead of antibodies, to bind to the target molecule, thyroxine (T4), is described. The DNAs which selectively bound to the T4 were selected, labeled with biotin or radioisotope, and then used to detection of T4 in the presence of liothyronine (T3), which has a chemical structure similar to that of T4. To synthesize binding assays that sense a target molecule, antibody production1 and molecular imprinting2 methods have been developed. In 1990, two research groups devised methods for in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX) for the production of oligonucleotide aptamers with high affinity toward target molecules.3,4 These oligonucleotide aptamers offer several potential advantages over traditional antibody-based reagents.5 They are not derived from living organisms and can be rapidly, reproducibly, and accurately synthesized by automated processes. In addition, labeling of oligonucleotides with various reporter molecules is simple and highly specific. Recently, some approaches using the DNA aptamers for diagnostic assay have been reported.5-7 These approaches usually cloned the selected oligonucleotides and used one of the clones. However, for some analytical purposes, it is not necessary to use monoclonal species. Noncloned aptamers that have different three-dimensional structures and bind to a target molecule at different binding sites could be used. These aptamers corresponded to polyclonal antibodies. We used the noncloned aptamers for chemical staining of a pattern-immobilized target molecule.8 †
NAIST. PRESTO. § Kyoto University. (1) Van Emon, J. M.; Lopez-Avila, V. Anal. Chem. 1992, 64, 79A-88A. (2) Kriz, D.; Ramstrom, O.; Mosbach, K. Anal. Chem. 1997, 69, 345A-349A. (3) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818-822. (4) Tuerk, C.; Gold, L. Science 1990, 249, 505-510. (5) Gold, L. J. Biol. Chem. 1995, 270, 13581-13584. (6) Drolet, D. W.; Moon-McDermott, Roming, T. S. Nat. Biotechnol. 1996, 14, 1021-1026. (7) Osborne, S.; Matsumura, I.; Ellington, A. D. Curr. Opin. Chem. Biol. 1997, 1, 5-20. (8) Kawazoe, N.; Ito, Y.; Imanishi, Y. Anal. Chem. 1996, 68, 4309- 4311. ‡
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In this note, in vitro selection of oligodeoxynucleotides for 3,5,3′,5′-tetraiodothyronine (L-thyroxine, T4), which is a hormone secreted from the thyroid gland and is clinically important to be diagnosed, was performed. The selected oligodeoxynucleotides were then used to assay T4 instead of polyclonal antibodies. MATERIALS AND METHODS Preparation of T3- and T4-Immobilized gels. T4-immobilized agarose gel was purchased from Sigma (St. Louis, MO). 3,5,3′-Triiodotyronine (liothyronine, T3) was immobilized on CNBractivated agarose gel (Pharmacia, Uppsala, Sweden) by the conventional method.9 In Vitro Selection. In vitro selection of DNA oligomers specific for T4 was carried out as described previously.10 For the first round of selection, synthetic 104-mer oligonucleotides with a random insert of 60 nucleotides, 5′-TAG-GGA-ATT-CGT-CGACGG-ATC-C-N60-CTG-CAG-GTC-GAC-GCA-TGC-GCC-G-3′ (primerbinding domains are underlined), were amplified using the primers 5′-TAA-TAC-GAC-TCA-CTA-TAG-GGA-ATT-CGT-CGA-CGG-AT3′ (P1) and 3′-GTC-CAG-CTG-CGT-ACG-CGG-C-5′ (P2). (The first 15 bases from the 5′-end of primer P1 correspond to the T7 RNA polymerase promoter region for the transcription reaction. This region is effective only for in vitro selection of RNA. Therefore, in the present study, the region has no meaning.) The synthetic ssDNA (∼2 µg, ∼109 molecules) was amplified by 10 polymerase chain reaction (PCR) cycles (one cycle: 94 °C, 15 s; 55 °C, 15 s; 72 °C, 15 s) in 100 µL of PCR reaction mixture [2.5 units of AmpliTaq DNA polymerase; 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 0.001% gelatin; 200 µM dNTPs; primers P1 (0.5 µM) and P2 (0.5 µM)]. The single-stranded (ss) DNA was then obtained from the double-stranded (ds) DNA by 30 additional asymmetric PCR cycles using only primer P1 [2.5 units of AmpliTaq DNA polymerase; 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 0.001% gelatin; 200 µM dNTPs; primer P1 (0.5 µM)]. The PCR-ssDNA was isolated by electrophorsis using 3% low-melting agarose gel (NuSieve GTG, FMC BioProducts, Rockland, ME) in order to remove unreacted primers and was purified (9) Affinity Chromatography; Pharmacia Biotechnology Co.: Uppsala, Sweden, 1994. (10) Ito, Y.; Suzuki, A.; Kawazoe, N.; Imanishi, Y. Bull. Chem. Soc. Jpn. 1997, 70, 695-698. S0003-2700(98)00081-X CCC: $15.00
© 1998 American Chemical Society Published on Web 06/26/1998
Figure 2. Dot blotting analysis of biotin-labeled DNA aptamers using Photogene kit with a streptavidin-alkaline phosphatase conjugate. Dots represents biotin-labeled DNAs that were eluted from the gel using T4 in the presence of T3 (O, A1-A3). The concentration of the eluted DNAs was determined by comparison with known concentrations of biotin-labeled DNA (P1-P4).
Figure 1. Schematic illustration of bioassay using DNA aptamers.
by a Qiaex II gel extraction kit (Hilden, Germany). The ssDNA pool (∼5 µg) was dissolved in 200 µL of binding buffer (0.5 M LiCl; 10 mM Tris-HCl, pH 7.6; 1 mM MgCl2), incubated at 95 °C for 5 min, and cooled over an hour. The annealed DNA pool was loaded onto a column packed with T4-immobilized gel column, incubated for 20 min at 15 °C, and subsequently rinsed with 1.2 mL of the binding buffer (six column volumes). The bound DNA was then eluted using three column volumes of distilled water at 15 °C. The eluted ssDNA (600 µL) was mixed with 100 mM MgCl2 (60 µL) and 3 mL of ethanol and incubated at -80 °C for 15 min to be precipitated. The precipitate was recovered by centrifugation (14000g) at 0 °C for 15 min, rinsed with 70% ethanol, and dissolved in 60 µL of distilled water. One-sixth of the ssDNA was amplified by PCR and used as the input DNA for the next round of selection. This process was repeated. The ability of the DNA strands to bind to the immobilized T4 was determined by comparing the amount of DNA loaded onto the column with that eluted from the column. The amount of
DNA was monitored by measuring the ultraviolet absorption at 260 nm. UV spectra were measured using a UV/visible spectrophotometer, Ubest-50 (Jasco, Tokyo, Japan). Competitive Binding Assay Using Biotin-Labeled DNAs. The 5′-ends of selected ssDNAs were labeled with biotin by PCR amplification performed as described previously.3 The DNAs (5 µg) were amplified by 30 cycles of PCR (one cycle: 94 °C, 15 s; 55 °C, 15 s; 72 °C, 15 s) in 2 mL of PCR mix [50 units of AmpliTaq DNA polymerase, 10 mM Tris-HCl, pH 8.3; 50 mM MgCl2: 0.001% gelatin; 200 µM dNTPs; 5′-biotin-labeled P1 primer (0.5 µM) instead of P1 primer]. A schematic of the binding assay procedure is shown in Figure 1. Biotin-labeled DNA aptamer was bound to T4-immobilized gel in buffered solution. The aptamer-bound gel (20 µL) was mixed with a sample solution (150 µL) containing various amounts of T3 and T4. After incubation for 20 min at room temperature, onethirtieth of the supernatant was removed and dot-blotted onto a nylon membrane (Biodyne transfer membrane, Pall, East Hills, NY). The concentration of biotin-labeled DNA aptamer was assayed using the Photogene (Life Technology, Rockville, MD) with a streptavidin-alkaline phosphatase conjugate. Competitive Binding Assay Using Radioisotope-Labeled DNAs. The selected ssDNAs were labeled by PCR amplification. The DNAs (5 µg) were amplified by 18 cycles of PCR (one cycle: 94 °C, 15 s; 55 °C, 15 s; 72 °C, 15 s) in the presence of [R-32P]dGTP (40 µCi) in 2.0 mL of PCR solution [50 units of AmpliTaq DNA polymerase, 10 mM Tris-HCl, pH 8.3; 50 mM MgCl2: 0.001% gelatin; 200 µM dNTPs; 0.5 µM P1 primer]. The labeled DNAs were purified by a QIA quick PCR purification kit (Hilden, Germany). A schematic of the bioassay procedure is the same as that using 32P-labeled DNA aptamer. The labeled aptamer, dissolved in 400 µL of binding buffer, was bound to T4-immobilized gel (0.8 mL) at 15 °C for 20 min. The aptamer-bound gel was rinsed with the binding buffer (10 times volume). A part of the aptamer-bound gel (50 µL) was mixed with a sample solution (200 µL) containing Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
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various amounts of T3 and T4. After incubation for 20 min at room temperature, the supernatant was collected and the radioactivity measured. RESULTS AND DISCUSSION The oligonucleotides with random sequences hardly adsorbed to the T4-immobilized gel. Even after the third round of selection, no significant bound DNA was detected. However, after the fifth round of selection, 17% of the applied DNA bound to the T4-immobilized gel. Consequently, 23% of the DNA bound to the gel after the seventh round of selection. There was a significant improvement in the ability of the oligonucleotides to bind to immobilized T4 as selection proceeded. The selected DNAs were labeled with biotin and then bound to T4-immobilized gel. Figure 2 shows the elution of biotin-labeled DNAs from T4-immobilized gel in the presence of soluble T4 (A1), soluble T3 and T4 (A2), or soluble T3 (A3). Known concentrations of biotin-labeled DNA were used for comparison (P1-P4). Comparing spot O and A1, A2, or A4, about 100 µM of DNAs was eluted by addition of T3 or T4 (the sizes and densities of dots were between P3 and P4). However, there was no significant difference in the amount of DNAs eluted between T3 and T4 (A3 vs A1). Therefore, “negative” selection was performed to improve the specificity to T4. In the negative selection, the DNAs from T4immobilized gel were applied in T3-immobilized gel, and nonbound DNAs were amplified by PCR. The process was repeated twice. Figure 3 shows the result for the elution of DNAs selected using T3-immobilized gel (negative selection). The DNAs was labeled with 32P-labeled nucleotides and bound to T4-immobilized gel. Subsequently, the DNAs were eluted from the gel in the presence of various concentrations of soluble T4 or soluble T3. DNAs were eluted by the addition of T4 but not by the addition of T3. The amount of eluted DNAs increased linearly with the increase of added T4. DNAs that specifically recognize T4 were selected, and the system can be used as an assay for T4. About
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Figure 3. Elution of DNA aptamer from the T4-immobilized gel in the presence of T4 (b) or T3 (O). Elution ratio (%) ) radioactivity of supernatant/total radioactivity (supernatant + gel) × 100. The bars represent the standard deviations; n ) 5.
1 molecule of DNA was eluted with the addition of 10 molecules of T4. Conventionally, polyclonal and monoclonal antibodies have been used for the detection of target molecules, dependent on the analytical purpose. Similarly, noncloned (polyclonal) DNA aptamers were used for assay in the present investigation. Considering that the aptamers are rapidly, reproducibly, and accurately synthesized by automated processes, and that they are simply and highly specifically labeled with various reporter molecules, this analytical procedure using DNA aptamers may replace the traditional antibody-based techniques. Received for review January 27, 1998. Accepted May 15, 1998. AC9800816
