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Bioconjugate Chem. 2001, 12, 850−854
In Vitro Selection of RNA Aptamers Carrying Multiple Biotin Groups in the Side Chains Yoshihiro Ito,*,† Akinori Suzuki,‡ Naoki Kawazoe,†,‡,§ and Yukio Imanishi§ Departmentof Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan, Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan, and Graduate School of Materials Science, NAIST, Ikoma 630-0101, Japan. Received November 2, 2000; Revised Manuscript Received April 30, 2001
In vitro selection or the systematic evolution of ligands by an exponential enrichment (SELEX) technique using a biotin-carrying nucleotide monomer was used for the development of a new type of molecular recognition sensor. Cytidine triphosphate (CTP) carrying the biotinyl group at the N4-position was applied for the technique. A pool of random sequence RNAs containing biotinyl groups in the side chains was prepared, and the RNAs binding to adenosine 5′-triphosphate (ATP) were selected. The selected nonnatural RNAs were used for an assay of ATP. Since they carried multiple biotin groups in the side chains, the sensitivity was high.
INTRODUCTION
Two methods, namely antibody production (1) and molecular imprinting (2), have been used to synthesize molecules sensing a target molecule. On the other hand, in vitro selection, also known as the systematic evolution of ligands by exponential enrichment (SELEX), was devised in 1990 for the identification of oligonucleotides, which are highly specific to target molecules. (3, 4) The selected oligonucleotides were named as aptamers and have been used as a new type of molecular sensor (5). These aptamers offer several potential advantages over traditional antibody-based reagents because they are not derived from living organisms and can be reproducibly, precisely, and quickly synthesized by automated processes. Recently, chemically modified nucleic acids were used in SELEX to expand the scope of the potential molecules (6-24). In the present study, cytidine 5′triphosphate carrying a biotinyl group at the N4-position {N4-[N-(N-biotinyl--aminocaproyl)-6-aminohexyl]cytidine 5′-triphosphate, Biotin-CTP} was subjected to the SELEX protocol, and a biotin-carrying RNA aptamer was synthesized and used as a new molecular recognition element. The molecular recognition element was expected to have a high sensitivity because it carried many biotin groups as probes. EXPERIMENTAL PROCEDURES
Chemicals. Biotin-CTP was purchased from the Life Technologies Co. (Rockville, MD). Custom synthesis of DNA oligomers was performed by the Sawady Technology Co. (Tokyo, Japan). AmpliTaq DNA polymerase was obtained from the Perkin-Elmer Co. (Foster City, CA). T7 RNA polymerase and RNasin were purchased from Promega Co. (Madison, WI). Dithiothreitol (DTT) was purchased from the New England Biochemicals Co. (Beverly, MA). [R-32P]GTP was purchased from the Am* Corresponding author: Fax/Tel: 81-88-656-7524. E-mail:
[email protected]. † University of Tokushima. ‡ Kyoto University. § NAIST.
ersharm Pharmacia Biotech. Co. (Buckinghamshire, UK). Reverse transcriptase Superscript II and a Photogene streptavidin/alkaline phosphatase conjugate were purchased from the Life Technologies Co. (Rockville, MD). ATP-immobilized gel and nucleoside 5′-triphosphates (NTPs) were purchased from the Sigma Co. (St. Louis, MO). Biodyne nylon membrane was purchased from the Pall BioSupport Co. (East Hills, NY). Transcription Reaction. A synthetic DNA pool containing a random region of 59 bases (5′-TAG-GGAATT-CGT-CGA-CGG-ATC-C-N59-CTG-CAG-GTC-GACGCA-TGC-GCC-G-3′) (80 ng) was amplified by 10 PCR cycles (94 °C, 15 s; 55 °C, 15 s; 72 °C, 15 s) in 800 µL of reaction solution containing Amplitaq DNA polymerase [Perkin-Elmer Co. (Foster City, CA)], 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 200 µM dNTPs, 0.5 µM 5′-primer (P1) containing the T7 promoter sequence (5′-TAA-TAC-GAC-TCA-CTA-TAGGGA-ATT-CGT-CGA-CGG-AT-3′), and 0.5 µM 3′-primer (5′-CGG-CGC-ATG-CGT-CGA-CCT-G-3′). The amplified template was precipitated with ethanol, and the resulting pellet was dissolved in 30 µL of RNase-free water. The DNA was transcribed with T7 RNA polymerase (4 µL, 1200 U) in a reaction mixture (100 µL) consisting of 80 mM Tris-HCl (pH 7.9), 12 mM MgCl2, 4 mM spermidine, 20 mM DTT, 20 µCi [R-32P] GTP, 0.4 U/µL RNasin, 1 mM of Biotin-CTP, and 1 mM other unmodified NTPs. This gave an approximate yield of 1 µg of random RNA. After incubation at 37 °C for 4 h, RQ1 RNase-free DNase (Promega, 2U) was added to the reaction solution to digest the DNA template. After incubation at 37 °C for 45 min, a small aliquot (10 µL) of the solution was analyzed by 7 M urea/8% denaturing polyacrylamide gel electrophoresis (PAGE). Dot blotting was carried out as described below. The transcripts were transferred to a Biodyne transfer membrane using the electroblotting method. The transcripts were then detected with a Photogene streptavidin-alkaline phosphatase conjugate according to the protocol provided by the manufacturer. Reverse Transcription. The transcription product (5 µL) was mixed with 5 µL RNase-free water and 2 µL
10.1021/bc0001342 CCC: $20.00 © 2001 American Chemical Society Published on Web 09/22/2001
RNA Aptamers Carrying Multiple Biotin Groups
of 10 µM 3′-primer P2. The mixture was incubated at 70 °C for 10 min and then chilled on ice. To this mixture were added 4 µL of a concentrated reverse transcription buffer (250 mM Tris-HCl, pH 8.3; 375 mM KCl; 15 mM MgCl2), 2 µL of 0.1 M DTT, and 1 µL of 20 mM dNTPs. Reverse transcriptase Superscript II (Life Technologies Co.) (200 U) was added to the mixture solution. After incubation at 42 °C for 50 min, the reaction was stopped by incubation at 70 °C for 15 min. Subsequently, the cDNA was amplified by PCR. Five microliters of the reverse-transcription reaction solution was added to a PCR solution (100 µL). After 10 PCR cycles on the same thermal condition as described, small aliquots (10 µL) were analyzed by 3% NuSieve GTG agarose gel electrophoresis. In Vitro Selection. The randomly sequenced RNA pool was prepared as described in the transcription experiment section and purified by denaturing PAGE on an 8% gel (100 mm × 100 mm in size, 2 mm thick). After excision of the band and elution into 0.6 mL of an elution buffer (0.5 M ammonium acetate; 10 mM magnesium acetate; 1 mM EDTA, pH 8.0; 0.1% SDS), the recovered RNA was precipitated with ethanol. The 32P-labeled RNA pool (1 µg) was dissolved in a binding buffer (0.3 M NaCl; 5 mM MgCl2; 20 mM Tris-HCl, pH 7.6; 5% DMSO; 0.5% Triton X-100), and the solution was incubated at 85 °C for 5 min. The solution was applied onto a column (0.5 mL) packed with an ATP-immobilized gel, and the solution and the gel were equilibrated for 30 min at 15 °C. Unbound RNAs were washed away with 40 column volumes of the binding buffer. The bound RNAs were eluted with six column volumes of a binding buffer containing 3 mM ATP and precipitated with ethanol in the presence of glycogen (200 µg). The resulting RNA pellet was dissolved in 30 µL of RNase-free water. The collected RNAs were amplified by RT-PCR, as described in the RT-PCR, and transcribed with [R-32P]GTP and Biotin-CTP, as described in the transcription experiments. The labeled RNA pool was again applied onto a column packed with ATP-immobilized gel, and bound RNAs were eluted. The amount of 32P-labeled RNAs was measured by Cerenkov counting. These processes were repeated. Binding Assay. The RNAs (1 µg) were added to the ATP-immobilized gel (0.1 mL). After washing with the binding buffer, 0.1 mL of GTP (3 mM), CTP(3 mM), UTP (3 mM), or ATP (0, 1, 2, 3 mM) was added to the gel to investigate the ligand specificity and the sensitivity for ATP. After incubation with the gel for 20 min, each of the eluted RNAs (5 µL) was spotted onto a Biodyne nylon membrane (1 cm × 1 cm). After each membrane dried for 15 min, it was irradiated with UV (365 nm) for 30 s and was incubated in 800 µL of blocking solution [3 wt % bovine serum albumin in TBS-Tween 20 buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05 v/v Tween 20)] at 65 °C for 1 h. Subsequently the membrane was incubated in TBS-Tween 20 buffer (800 µL) containing streptavidin-alkali phosphatase (0.083 µg/mL, GIBCO) for 10 min at room temperature. The membrane was washed with the TBS-Tween 20 buffer twice (800 µL × 2), was placed in a microplate well, and was incubated with a final buffer (800 µL, 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2) containing 10 mM p-nitrophenyl phosphate for 1.5 h at 37 °C. The absorbance (405 nm) of supernatant of each well was measured, and the amount of eluted RNAs was determined.
Bioconjugate Chem., Vol. 12, No. 6, 2001 851
Figure 1. 8% Denaturing PAGE analysis of the transcription products (10 µL) at various concentrations of modified BiotinCTP and CTP. The gel was stained by ethidium bromide. The faint band at around a 100 base length corresponds to the DNA template.
Figure 2. Dot blotting of the transcription products in the presence of Biotin-CTP or CTP. The biotin residue is detected with a Photogene kit.
Figure 3. Agarose gel electrophoresis analysis of RT-PCR products from the transcripts containing Biotin-CTP. RESULTS AND DISCUSSION
For each round, the SELEX process requires the transcription of RNA, the isolation and reverse transcription of the target-bound RNA, and PCR amplification of the cDNA. To introduce modifications to RNA, a modified nucleotide triphosphate must be a substrate for T7 RNA polymerase. The modified RNA, on the other hand, must be a substrate for reverse transcriptase for cDNA synthesis. The use of Biotin-CTP in place of CTP in the transcription reaction gave biotin-incorporated transcripts (Figure 1). The biotin-incorporated transcripts migrated more slowly in gel electrophoresis than normal transcripts. The yield of biotin-modified transcripts was lower than that of normal transcripts. The presence of the biotinyl group in the biotin-incorporated transcripts was confirmed by dot blotting (Figure 2). Another requirement for the SELEX protocol is that the modified RNA must serve as a template for reverse transcriptase to produce cDNA. When the biotin-incorporated transcripts were reverse-transcribed to cDNA, they gave the full-length of cDNA (Figure 3). The band density increased with the increase of the PCR cycle. Comparing the efficiency of the reverse transcription reaction in the presence of Biotin-CTP with that of CTP, both were almost the same. The sequential analysis of the product cDNA demonstrated that the cDNA was the same as that of the original synthetic DNA.
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Figure 5. Binding affinity of RNAs transcribed from BiotinCTP or CTP. Figure 4. Secondary structure of cloned RNAs. The symbols - and • represent Watson-Crick base-paring and Wobble basepairing, respectively. Biotin-CTP was used instead of CTP.
These results demonstrated that Biotin-CTP is applicable to the SELEX experiment, although the bulky biotin group existed. In Vitro Selection. The selection of aptamers for ATP was started from a biotin-modified random sequence pool of RNA molecules, which were prepared by transcription reaction using synthetic DNA with random sequences as templates. The diversity was theoretically considered to be 1012-1013 according to the estimation by Ellington and Szostak (3). The transcripts were added to the ATPimmobilized agarose gel. After being washed with the binding buffer, the bound RNA molecules were eluted using soluble ATP. The collected RNA molecules were reverse-transcribed and amplified by PCR. The amplified cDNAs were again transcribed to RNA molecules using Biotin-CTP again, and the transcripts were added to ATP-immobilized gel. These processes were repeated. At the final round, counter-SELEX was carried out using soluble GTP. When measuring the binding ratio of RNA to ATP-immobilized gel by dividing the amount of RNA molecules eluted by soluble ATP with that of all RNA molecules added, it was found that the ratio increased with the increase of the selection round (1st, 0%; 2nd, 3%; 3rd, 7%; 4th, 7%; 5th, 8%; 6th, 12%; and 7th, 31%). After eight rounds of selection process, 42% of RNA molecules were found to bind to the ATP-immobilized gel. At this point, PCR-amplified DNA was cloned, and 32 clones were sequenced, resulting in only two different sequences (Figure 4). The sequential variety of the selected biotin-modified RNA, which was lower than that of normal RNA, might be explained in terms of the lower yield of transcription products of biotin-modified RNA as compared with that of normal RNA. Both of the biotinmodified RNAs had approximately 10% of the Biotin-CTP component. This result indicated that the biotin residue suppressed the incorporation of Biotin-CTP in the transcripts. The requirement of the biotin residues for affinity binding was tested by synthesizing normal transcripts with unmodified CTP. The transcripts did not bind to the ATP-immobilized gel (Figure 5). This result suggested that the presence of the biotin groups was critical for the high affinity of the biotin-modified RNA. The biotin groups should contribute to adopt conformations suitable for recognizing ATP or to interact directly with ATP. After selected RNAs were bound on the ATP-immobilized gel, known concentrations of nucleotide triphos-
Table 1. Elution Percentage of RNAs Bound to ATP-Immobilized Gel by Nucleotide 5′-Triphosphatesa noncloned clone 1 clone 2 a
ATP
CTP
UTP
GTP
100 100 100
10 7 2
7 5 4
20 7 3
The percentage of RNAs eluted by ATP was taken as 100%.
phate were added to elute the RNAs, and the eluted RNAs were measured by conventional biotin-avidin interaction (Table 1). The selected RNAs were eluted only by ATP. It was indicated that the triphospate region is not important for the recognition of RNAs. When adenosine monophosphate (AMP), adenosine diphosphate (ADP), or 3′-deoxyadenosine triphosphate (dATP) was added to the clone 2 bound to ATP-immobilized gel, the clone was eluted (ADP, 85%; AMP, 90%; and dATP, 80%). These results indicated that the RNAs recognized the ATP at the base moiety. When streptavidin was added to the clone 2 bound to the ATP-immobilized gel, no significant amount of clone was eluted from the gel. However, the biotin of the clone could be detected with a conventional biotin-streptavidin detection kit. It was considered that the bound clone had a folded structure to inhibit streptavidin from invading the biotin residue in the binding state but that the biotin of the eluted clone binds the streptavidin after the denaturation (unfolding) for detection (Figure 6). The dissociation constants of both clones 1 and 2 were 10 and 15 µM, respectively. The dissociation constants of normal RNA or DNA selected for ATP ranged from 14 to 6 µM according to the reports of Sassanfar and Szostak (25) and Huizenga and Szostak (26), respectively. The dissociation constants of biotin-carrying RNA aptamers in the present study were almost the same order of those of normal oligonucleotide aptamers. Dieckmann et al. (27) reported that several highly conserved nucleotides in the binding pockets could be substituted while retaining binding under NMR conditions. Figure 7 shows the result of sensing the concentration of ATP by clone 2. The clone 2 RNA bound to the ATP-immobilized gel was released from the gel with the increase of ATP. This result demonstrates that the selected RNAs quantitatively recognize ATP and can therefore be used as a molecular sensor for ATP. All of the six biotin residues on the aptamer were not recongnized by the streptavidin. The sensitivity of the assay using the aptamer carrying multiple biotin residues had about three times as much as that labeled with single biotin. Considering the development of a new biotin detection system (28), the
RNA Aptamers Carrying Multiple Biotin Groups
Bioconjugate Chem., Vol. 12, No. 6, 2001 853 ACKNOWLEDGMENT
Y. Ito expresses his appreciation for the financial support received as a grant-in-aid (number 09878117) from the Ministry of Education, Science, Culture, and Sports of Japan. LITERATURE CITED
Figure 6. Schematic drawing of interaction of biotin-carrying RNA aptamer with target molecule, ATP. Streptavidin did not bind the biotin of aptamer bound to the ATP-immobilized gel but bound to the eluted aptamer.
Figure 7. Binding assay of ATP concentration by using aptamers carrying multiple biotin groups in the side chains. n ) 4. Bars represent standard deviations.
aptamers carrying multiple biotin groups will offer a new detection system with higher sensitivity. Recently, aptamers have been used for various analytical purposes (7). The present study develops a new methodology to synthesize a chemosensor instead of using antibodies. For biotin-carrying RNAs, it is a great advantage not to have to worry about denaturation by the labeling or modification process in comparison with antibodies. In addition, these RNAs carry a number of probes in a chain to increase sensitivity in comparison with other aptamers consisting of natural nuleotides. Taking into consideration the convenience of RNA preparation in vitro, this type of RNA sensor will replace the traditional antibody sensors, which are produced from animals.
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