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Tailing DNA aptamers with a functional protein by two-step enzymatic reaction
Journal of Bioscience and Bioengineering VOL. 116 No. 6, 660e665, 2013 www.elsevier.com/locate/jbiosc
Tailing DNA aptamers with a functional protein by two-step enzymatic reaction Mari Takahara,1 Kounosuke Hayashi,1, 3 Masahiro Goto,1, 2 and Noriho Kamiya1, 2, * Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan,1 Center for Future Chemistry, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan,2 and Hitachi Aloka Medical, Ltd., 6-22-1 Mure, Mitaka-shi, Tokyo, Japan3 Received 22 April 2013; accepted 15 May 2013 Available online 25 June 2013
An efficient, quantitative synthetic strategy for aptamer-enzyme conjugates was developed by using a two-step enzymatic reaction. Terminal deoxynucleotidyl transferase (TdT) was used to first incorporate a Z-Gln-Gly (QG) modified nucleotide which can act as a glutamine donor for a subsequent enzymatic reaction, to the 30 -OH of a DNA aptamer. Microbial transglutaminase (MTG) then catalyzed the cross-linking between the Z-QG modified aptamers and an enzyme tagged with an MTG-reactive lysine containing peptide. The use of a Z-QG modified dideoxynucleotide (Z-QG-ddUTP) or a deoxyuridine triphosphate (Z-QG-dUTP) in the TdT reaction enables the controlled introduction of a single or multiple MTG reactive residues. This leads to the preparation of enzyme-aptamer and (enzyme)n-aptamer conjugates with different detection limits of thrombin, a model analyte, in a sandwich enzyme-linked aptamer assay (ELAA). Since the combination of two enzymatic reactions yields high site-specificity and requires only short peptide substrates, the methodology should be useful for the labeling of DNA/RNA aptamers with proteins. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Enzyme-linked aptamer assay; DNA aptamer; DNA-enzyme conjugate; Microbial transglutaminase; Thrombin; Terminal deoxynucleotidyl transferase]
Nucleotide aptamers are nucleic acids that are able to bind target molecules with high affinity and specificity, which make them promising biorecognition elements (1,2). Compared with general receptor molecules such as antibodies, aptamers have several advantages as nucleic acids (3). Their in vitro production (SELEX; systematic evolution of ligands by exponential enrichment) (2) allows easy chemical synthesis, modification, and high stability after modification. These unique properties suggest their potential for applications in therapeutics, diagnosis, and analysis (3e6). Labeling of aptamers with functional molecules is important for practical applications. For signal amplification in molecular diagnostics (5), many DNA aptamer-enzyme conjugates have recently been developed using non-covalent interactions such as the biotineavidin interaction (7,8). Particular properties of DNA aptamers, such as high target specificity and affinity, render these molecules potential candidates for functionalization of nanomaterials (9). Thus, introducing target specificity of aptamers to nanomaterials can extend the application of aptamer-nanomaterial hybrid to intracellular analysis, cancer cell imaging and drug delivery. In order to maximize the functions of aptamer-enzyme/nanomaterial conjugates, site-specific labeling is required. For site-specific covalent linking of aptamer and enzyme, we herein propose a novel two-step tailing method using terminal deoxynucleotidyl
* Corresponding author at: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan. Tel.: þ81 92 802 2807; fax: þ81 92 802 2810. E-mail addresses: [email protected], nori_kamiya@ mail.cstm.kyus (N. Kamiya).
transferase (TdT) (10e13) and microbial transglutaminase (MTG) (14,15). The label was introduced by enzymatic reaction specifically to the 30 -OH termini of aptamers, enabling the preparation of bifunctional aptamer-enzyme conjugates. The aptamer tailing was limited by the ability of the DNA/ RNA polymerase to incorporate modified deoxynucleoside triphosphates (6). In addition, template-independent tailing is necessary because aptamers recognize target molecules in a single strand (1,2), but most polymerases requires both template nucleic acids and a primer (16). There is an unusual polymerase, TdT, which is able to catalyze template-independent addition of nucleotides to the 30 -OH termini of double- and single-stranded nucleic acids (16). TdT also incorporates modified nucleotides which leads to terminal labeling of certain molecules such as biotin and digoxigenin (17e22). However the sizes of enzyme-modified nucleotides are too large for TdT to recognize as substrates. Instead, we used MTGmediated cross-linking between modified aptamers and enzymes, and TdT for introducing MTG-reactive substrates to aptamers. MTG catalyzes a substrate specific acyletransfer reaction between the ε-amino group in lysine (Lys) and the g-carboxamide group in glutamine (Gln) (23). The use of MTG is crucial to maximize both the functions of aptamers and the labeled enzyme. DNA-protein conjugates (24,25) are usually synthesized by chemical modification and it depends on the functional groups of the target proteins. Random orientation of the reactive groups on the protein surface yields heterogeneous chemical conjugates with partial loss of function. By taking advantage of the high site-specificity of MTG, these problems are avoided, enabling attachment at the desired position (26). In our
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.05.025
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previous research, we synthesized an acyl-donor nucleotide substrate, 5-[3-(Z-Gln-Gly-amido)-1-(E)-propenyl]-20 -deoxyuridine 0 5 -triphosphate (Z-QG-dUTP), and incorporated it into DNA using PCR (27). In parallel, an acyl-acceptor Lys-containing tag (K-tag) sequence was fused to the N-terminus of the target protein, and site-specific labeling of Z-QG containing DNA using a K-tagged enzyme was achieved. In this study, we describe the use of a combination of TdT and MTG for site-specific tailing of a DNA aptamer with single or multiple enzymes. Thrombin was selected as a model analyte in order to validate the function of the enzyme-aptamer conjugates obtained. TdT incorporated Z-QG modified nucleotides into thrombin binding aptamers, and Z-QG labeled aptamers were conjugated with K-tagged bacterial alkaline phosphatase (BAP) using MTG (Fig. 1). The number of labeled BAPs can be controlled by using Z-QG-dUTP or Z-QG-ddUTP, a dideoxynucleotide analog, as terminators of the TdT reaction (18,19,28). The obtained BAP-aptamer conjugates were applied in a sandwich enzyme-linked aptamer assay (ELAA) (29e32) for thrombin to analyze both the functions of the aptamers and the labeled enzymes. In the sandwich ELAA, the 29-mer thrombin-binding aptamer (33) was immobilized as the primary aptamer, and the 15-mer thrombin-binding aptamer (34) modified by the two-step enzymatic reaction was the secondary aptamer. MATERIALS AND METHODS Chemicals Recombinant terminal deoxynucleotidyl transferase (TdT), and NBT/BCIP stock solution were purchased from Roche Applied Science (Basel, Switzerland). Z-QG-dUTP, Z-QG-ddUTP, 5-[3-(15-Z-Gln-Gly-amido-4,7,10,13-tetraoxapentadecanoylamido)-1-(E)-propenyl]-20 -deoxyuridine 50 -triphosphate (Z-QG-TEOdUTP), and 5-[3-(15-Z-Gln-Gly-amido-4,7,10,13-tetraoxapentadecanoylamido)-1-(E)-
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propenyl]-20 ,30 -deoxyuridine 50 -triphosphate (Z-QG-TEO-ddUTP) (Fig. S1) were synthesized at Gene Act, Inc. (Fukuoka, Japan). MTG was supplied by Ajinomoto Co., Ltd. (Tokyo, Japan) and was dissolved in 5 mM TriseHCl (pH 8.0). FX174 DNA-HaeIII Digest was purchased from New England Biolabs Ltd. (Ontario, Canada). Agarose S, albumin from bovine serum, Cohn fraction V, pH 7.0 (BSA), boric acid, magnesium chloride hexahydrate (99.9%), N-ethylmaleimide (NEM), sodium dodecyl sulfate (SDS) and tris (hydroxymethyl)aminomethane were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Formamide, potassium chloride, sodium chloride and urea were purchased from Kishida Chemical (Osaka, Japan). Tween20 was purchased from SigmaeAldrich (St. Louis, MO, USA). The Nunc Immobilizerstreptavidin plate (SA plate) was purchased from Nalge Nunc International Co. Ltd (Tokyo). The ECF substrate solution was purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK). Thrombin from human plasma was purchased from Merck Ltd. (Darmstadt, Germany). The thrombin binding aptamers (Tsukuba Oligo Service Co. Ltd., Tsukuba, Japan) used in this study have the following sequence: fluorescein isothiocyanate (FITC)emodified 15-mer aptamer with polyT15 tail, 50 -FITC-GGT TGG TGT GGT TGG TTT TTT TTT TTT TTT-30 ; biotinylated 29-mer aptamer with polyT20 spacer, 50 -biotin- TTT TTT TTT TTT TTT TTT TTA GTC CGT GGT AGG GCA GGT TGG GGT GAC T-30 . The underlined sequence, 50 - GGT TGG TGT GGT TGG-30 was reported by Bock et al. (34), and another sequence, 50 -AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-30 was reported by Tasset et al (33). These two thrombin binding aptamers have different binding sites for thrombin with Kds of 26 nM and 0.5 nM, respectively. Preparation of recombinant BAP A K-tagged BAP was prepared according to our previous reports. A K-tag sequence, MKHKGGGSGGGSGS (the underlined K is the MTG reactive residue) was fused to the N-terminus of BAP. The resultant BAP was abbreviated as NK14-BAP (35). The NK14-BAP gene was inserted into the pET22 (þ) plasmid vector. The recombinant NK14-BAP was produced using Escherichia coli BL21(DE3). The crude protein extract was purified using a HisTrap HP column 5 mL, and PD10-column (GE Healthcare) according to the manufacture’s instruction. The eluted protein was then purified using a size-exclusion chromatography (SEC) column equilibrated with 5 mM TriseHCl (pH 8.0). A SEC column was constructed by packing the XK16 column with Superdex 200 prep grade that were both purchased from GE Healthcare). All the chromatography experiments for the purification of NK14-BAP were conducted on a BioLogic DuoFlow Chromatography System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Purified NK14-BAP was analyzed by SDS-polyacrylamide gel electrophoresis (SDSPAGE) stained using Bio-Safe Coomassie (Bio-Rad Laboratories, Inc.) (Fig. S2). The
FIG. 1. Tailing aptamer with K-tagged BAP by a two-step enzymatic reaction. TdT incorporated Z-QG molecules into aptamers. MTG catalyzed cross-linking between Z-QG aptamers and K-tagged BAP (NK14-BAP). TdT needs only single strand DNA and nucleotides, which allows the use of MTG without purification of the primer.
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calculated molecular weight of NK14-BAP was 50 kDa. The protein concentration was determined with a Bicinchoninic Acid Protein Assay Kit (Thermo) using bovine serum albumin as a standard. Tailing aptamers with Z-QG modified nucleotides The Z-QG labeling reaction was conducted in a total volume of 100 mL by mixing the following compounds on ice: aptamer (FITC modified 15-mer), 5 mM; CoCl2, 5 mM; Z-QG-(TEO)ldUTP solution, 0.5 mM; or Z-QG-(TEO)l-ddUTP solution, 0.05 mM (l ¼ 0, 1); and 400 U/reaction TdT in reaction buffer (potassium cacodylate, 200 mM; TriseHCl, 25 mM; and BSA, 0.25 mg/mL, pH 6.6). The reaction solutions were incubated at 37 C for 2 h and terminated by heating at 94 C for 15 min. The TdT reaction products were directly subjected to denaturing polyacrylamide gel electrophoresis (PAGE) and analyzed by visualizing their fluorescently modified 50 -end. Denaturing PAGE A total of 1 mL TdT reaction products was diluted with ultra pure water to 5 mL then mixed with 5 mL formamide and 1 mL loading buffer. The denatured TdT reaction mixtures were subjected to electrophoresis using 15% denaturing gel containing 1 TBE buffer and 7 M urea at 280 V for 40 min. The gel was imaged with a fluorescent imager (Molecular Imager FX Pro, Bio-Rad Laboratories, Inc.) with an excitation wavelength of 488 nm and a 530 (15) nm band pass filter. The reaction products were visualized by their 50 -FITC (excitation wavelength of 488 nm and emission wavelength of 530 nm). Labeling Z-QG aptamers with NK14-BAP The TdT reaction solutions were subjected to a Probe Quant G50 spin column (GE Healthcare) to remove nonextended aptamers and unreacted Z-QG-(TEO)l-dUTPs or Z-QG-(TEO)l-ddUTPs. After purification, Z-QG aptamers were labeled with NK14-BAP using MTG. The MTG reaction mixture (300 mL) consisted of Z-QG aptamer (0.5 mM), NK14-BAP (2.5 mM; (ZQG-(TEO)l)m-aptamer, 1.25 mM; Z-QG-(TEO)l-aptamer), and MTG (0.1 U/mL) in 20 mM TriseHCl buffer (pH 7.4). The MTG reaction solutions were incubated at 4 C for 3 h and terminated by NEM (1 mM). The obtained BAP-aptamer conjugates were analyzed using 2.0% agarose gel electrophoresis in 1 TBE buffer. Electrophoresis was conducted at 135 V for 27 min. The gel was imaged using a fluorescent imager for aptamers as described previously. Ultimately, the gel was stained in NBT/BCIP solution following an established protocol below. NBT/BCIP coloring reaction In the coloredevelopment reaction, NK14-BAPcatalyzed dephosphorylation of 5-bromo-4-chloro-3-indolyl phosphate (BCIP), thereby priming the agarose gel for the blue precipitates to appear on the agarose gel following reduction of p-nitroblue tetrazolium chloride (NBT). NBT/BCIP stock solution (18.75 mg/mL NBT and 9.4 mg/mL BCIP) was dissolved in NTMT buffer (100 mM NaCl, 100 mM TriseHCl, 50 mM MgCl2, pH 9.5). The gel was incubated in prepared NBT/BCIP solution at 37 C for 40 min. General enzyme-linked aptamer assay (ELAA) protocol This protocol is based on our previous research. All chemicals were diluted with Tris Buffered Saline (TBS; 25 mM TriseHCl, 137 mM NaCl, 2.68 mM KCl, pH 7.4), unless specified. All the steps were carried out at 37 C. The SA plate was washed three times with TBST (TBS containing 0.05% v/v Tween20) after the addition of each biomolecule. Before the immobilization, the biotinylated 29-mer aptamer was heated at 94 C for 15 min to anneal the DNA strand and then cooled on ice for 10 min in order to keep the DNA structure unfolded. The biotinylated 29-mer aptamer (200 nM, 100 mL) was immobilized on the SA plate. After incubation for 1 h and washing, thrombin (100 nM, 100 mL) was added to the SA plates. After incubation for 1 h and washing, the BAP-aptamer conjugate (50 nM, 100 mL) was added to the SA plate. After incubation for 1 h and washing, ECF substrate (1 mM, 100 mL) was added to the SA plate. The ECF substrate was diluted with NTMT buffer. After incubation for 30 min, the enzymatic product was detected by an LS-55 Fluorescence Spectrometer (PerkinElmer) using the fluorescence intensity at 560 nm with an excitation wavelength of 430 nm.
RESULTS AND DISCUSSION Tailing aptamers with Z-QG modified nucleotides TdT adds a nucleotide or nucleotide analog to the 30 -OH end of singlestranded DNA. To introduce Z-QG molecules, Z-QG-ddUTP and ZQG-dUTP were employed in a TdT-catalyzed tailing reaction. The dideoxynucleotide analogue was prepared for the single Z-QG labeled aptamer because TdT initiates homopolymerization by recognizing the 30 -OH end of nucleic acids. We also focused on the effect of polyethylene oxide (PEO) linker on the TdT reaction by introducing Z-QG-TEO-ddUTP and Z-QG-TEO-dUTP. The model aptamer was a thrombin-binding 15-mer aptamer, and a 50 -FITC modified 15-mer aptamer with polyT15 tail was designed for the TdT reaction (36). Fig. 2 shows the result of denaturing PAGE analysis of the TdT reaction products. Lanes 2e5 (TdT reaction products) showed the uniform mobility and the disappearance of the original aptamer band (lane 1). This result suggests all aptamers were labeled with
FIG. 2. Denaturing PAGE analysis of the TdT reaction products. In the TdT reaction mixture, no nucleotides (lane 1), Z-QG-ddUTP (lane 2), Z-QG-TEO-ddUTP (lane 3), ZQG-dUTP (lane 4), and ZQG-TEO-dUTP (lane 5) were incubated with aptamers and CoCl2. M: FX174 DNA-HaeIII digest treated with FITC-ddUTP, CoCl2, and TdT.
the Z-QG moiety and TdT can recognize all the Z-QG modified ddUTP/dUTPs as substrates. Namely, perfect conversion of aptamers was achieved in the TdT reaction regardless of Z-QG and PEO modifications. Z-QG-dUTP labeled aptamers were shifted to higher mobility than Z-QG-ddUTP labeled ones because of the addition of several Z-QG molecules (lanes 2, 3 and lanes 4, 5, respectively). The average number of labeled Z-QG was estimated by the absorbance of 296 nm (27). Incubation with the dideoxynucleotide analogs (ZQG-ddUTP or Z-QG-TEO-ddUTP) yielded 1:1 stoichiometric labeling of the aptamer with single Z-QG moiety. On the other hand, the number of Z-QG moiety per aptamer was calculated to be approximately two for Z-QG-dUTP and three for Z-QG-TEO-dUTP, respectively. The resultant Z-QG multi-labeled aptamers (i.e., (ZQG)m-aptamer and (Z-QG-TEO)m-aptamer) showed a slower mobility shift than Z-QG single-labeled aptamers. The PEO linker effect is shown in lane 5. TdT polymerizes nucleotides randomly therefore the reaction product is characterized by various tail lengths and appears as a broad band in Fig. 2. (ZQG)m-aptamers were a broad band because of random addition (Fig. 2, lane 4), but (Z-QG-TEO)m-aptamers resulted in narrower size distribution (Fig. 2, lane 5), associated with the PEO linker effect. The efficiency of the TdT reaction depends on the type of nucleotides that affect the structure of the 30 -termini of DNA/RNA. The effect of the PEO linker is not yet fully understood, but multiple labeling of Z-QG might increase the hydrophobicity of the 30 termini of the aptamer resulting in an inability of TdT to access the sterically hindered 30 -OH end. The introduction of the PEO linker may reduce this assumed effect and thus allow a more controlled mean tail length. Labeling Z-QG aptamers with NK14-BAP Transglutaminase catalyzes the acyl transfer reaction between the ε-amino group in Lys and the g-carboxamide group of Gln in peptides and proteins (37,38). Microbial transglutaminase from Streptomyces mobaraensis (MTG) can catalyze conjugation of a K-tagged enzyme and Z-QG modified DNA in a Ca2þ independent manner (14). Z-QG is a neutral dipeptide substrate recognized by MTG as an acyl-donor. A recombinant protein, K-tagged BAP (NK14-BAP), has an acylacceptor tag sequence (K-tag) at the N-terminus (35). We examined the site-specific labeling of Z-QG aptamers with NK14BAP using MTG. Fig. 3 shows the agarose gel electrophoresis analysis of the obtained BAP-aptamer conjugates. Each lane shows the mobility shifts and the disappearance of the original Z-QG aptamer bands. Since few residual Z-QG aptamers were observed, almost all Z-QG labeled aptamers were converted to NK14-BAP-aptamer conjugates in the MTG catalyzed reaction (Fig. 3a). As shown in Fig. 3b, NK14-BAP was stained by the BAP-catalyzed NBT/BCIP coloring reaction. The mobility shifts of NK14-BAP
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FIG. 3. Agarose gel electrophoresis analysis of MTG reaction products. (a) DNA aptamers were imaged with FITC, and (b) NK14-BAP was detected by the BAP-catalyzed NBT/BCIP colouring reaction in the same gel. Lane 1: aptamer alone. A mixture of NK14-BAP and Z-QG-aptamer (lanes 2 and 4), Z-QG-TEO-aptamer (lanes 3 and 5), (Z-QG)m-aptamer (lanes 6 and 8) or (Z-QG-TEO)m-aptamer (lanes 7 and 9) was incubated without MTG (lanes 2, 3, 6, and 7) or with MTG (lanes 4, 5, 8, and 9). M: FX174 DNA-HaeIII Digest.
corresponded to that of the aptamers. This observation supports the mobility shift derived from labeled NK14-BAP. Conjugated BAP showed higher mobility than unconjugated BAP because of the negative charge of aptamers. The different mobility in electrophoresis could be determined by their charge differences. Similar results were reported in the case of a thrombin-aptamer complex (31). The Z-QG multi-labeled aptamers were shifted to lower mobility compared to the Z-QG single-labeled aptamers because of multiple labeling of NK14-BAP. In addition, MTG can catalyze efficient cross-linking in the presence of PEO linker (Fig. 3b, lanes 5 and 9). From quantitation of incorporated Z-QG, multiple labeled aptamer would have at most 2- or 3-labeled BAPs, resulting in high BAP activity in NBT/BCIP coloring reaction (Fig. 3b, lanes 8 and 9). In contrast, low BAP activity of single-labeled aptamer was observed (Fig. 3b, lanes 4 and 5). The lower activity was resulting from the difference of conjugated number of BAP. By the two-step enzymatic reaction, preparation of a single-enzyme labeled aptamer (i.e., BAPaptamer) and a multiple-enzyme labeled aptamer (i.e., (BAP)naptamer) was successfully conducted. The above described enzymatic strategy should prove useful for the development of various aptamer-protein conjugates because the combination is not limited in this system and quantitative conversion is achieved. MTG requires only short reactive peptides containing Gln in the aptamer and Lys in the recombinant protein. The Z-QG is easily introduced into the aptamer in the TdT reaction using Z-QG modified nucleotides. ELAA with BAP-aptamer conjugates BAP-aptamer conjugates labeled by a two-step enzymatic reaction were then applied to the sandwich ELAA for thrombin (Fig. 4a). The sandwich assay was performed using streptavidin-coated 96-well microplates (SA plates). The biotinylated 29-mer aptamer (200 nM) was immobilized on the SA plates as a primary aptamer, and thrombin (100 nM) was added. After thrombin capture, a BAPaptamer conjugate (50 nM) employed as a secondary aptamer was added for detection. The SA plates were washed between each step to remove residual compounds. The conjugate concentrations were optimized in accordance with the response of BAP-aptamer (Fig. S3). Both functions of aptamer and BAP were evaluated by measuring the relative activity of BAP. First, we investigated the nonspecific reaction of the BAPaptamer conjugates. The biotin-streptavidin system, one of the most general (non-covalent) tailing methods, is broadly used because of its natural high affinity. However this strong interaction also causes a high level of nonspecific reaction leading to falsepositive results. Since the BAP-aptamer conjugates in this study were covalently tailed, the nonspecific reaction could be minimized. As shown in Fig. 4b, in the presence of all components
(Fig. 4b, lane 5), the (BAP)n-aptamer showed increased relative activity although a very low BAP activity was observed in the absence of each component (Fig. 4b, lanes 1e3). The BAP-aptamer showed lower activity compared with the (BAP)n-aptamer. The decreased activity from the single labeling made it difficult to distinguish between specific and nonspecific binding. We also examined the selectivity of BAP-aptamer conjugates by mixing other protein (BSA) instead of thrombin. Little activity was observed as shown in Fig. 4b, lane 4. These results confirmed that BAP-aptamer conjugates selectively bind to thrombin without nonspecific adsorption especially for the (BAP)n-aptamer. In the presence of thrombin, a 4-fold increase in activity of (BAP)naptamers to BAP-aptamers was observed. This difference should result from the number of labeled BAP or the effect of labeled BAP on binding affinity. To study the dynamic range and sensitivity of the assay using labeled aptamers, BAP-aptamer conjugates were incubated with thrombin at various concentrations in the range of 0e100 nM. A doseeresponse curve for thrombin was carried out (Fig. 5). The curve was fitted by sigmoidal regression using the following four-
FIG. 4. ELAA applications. (a) Scheme of the sandwich ELAA. (b) Relative BAP activities of BAP-aptamer conjugates under various conditions. The immobilized aptamer on the plate captured thrombin, then the BAP-aptamer conjugate bound to thrombin (lane 5). As a negative control, ELAA was performed in the absence of BAP-aptamer conjugate (lane 1), biotinylated aptamer (lane 2) and thrombin (lane 3). Instead of thrombin, BSA was utilized (lane 4). The BAP activities were relative to the value obtained in the presence of biotinylated aptamer, thrombin and BAP-aptamer (lane 5, white histogram). All error bars represent standard deviations obtained through the detection of three parallel samples. Open bars, BAP-aptamer; closed bars, (BAP)n-aptamer. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons within all lanes. *p < 0.05 versus lanes 1e4 for each conjugate.
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J. BIOSCI. BIOENG., biomolecules on a solid surface was shown to decrease their functions as a result of their limited movement (42). The PEO linker provides labeled enzymes flexibility, which could increase enzymatic activity at a solid surface. The dynamic range of the BAPTEO-aptamer was 1e25 nM and its DL was 0.36 nM. Since an improved result was obtained by using the BAP-TEO-aptamer, we also investigated the (BAP-TEO)n-aptamer. The dynamic range of the (BAP-TEO)n-aptamer was 1e100 nM and its DL was 0.65 nM (Fig. S4). The introduction of the PEO linker to the BAP multilabeled aptamer resulted in a wider dynamic range but an improvement in the DL was not observed.
FIG. 5. Doseeresponse curve for thrombin. ELAA was performed under different concentrations of thrombin in the range of 0e100 nM. The BAP activities were relative to the value obtained in the presence of thrombin (100 nM) and BAP-aptamer. All error bars represent standard deviations obtained through the detection of three parallel samples. Open circles, BAP-aptamer; open triangles, BAPeTEO-aptamer; closed circles, (BAP)n-aptamer.
parameter logistic equation with KaleidaGraph (Synergy Software, Reading, PA, USA) (7,39,40): y ¼ dþ
ad 1 þ ðx=cÞb
(1)
Conclusions Tailing aptamers with K-tagged enzymes by a two-step reaction was achieved. The two-step reaction was useful to modify all aptamers, providing 100% conversion. Because of its template-independency and its activity toward a wide range of substrates, TdT is an efficient polymerase for tailing DNA aptamers. MTG can catalyze site-specific cross-linking between Z-QG modified aptamers and K-tagged proteins which avoids inactivation of both functions. The functions of both aptamers and enzymes were confirmed using ELAA analysis. TdT can incorporate mono or several modified nucleotides to the 30 -OH end of nucleic acids. This is a controlled single- or multi-labeling reaction compared to random chemical conjugation. Even in the presence of PEO modification, TdT and MTG can recognize the aptamers as substrates, and the two-step reaction proceeds efficiently. The two-step reaction catalyzed by TdT and MTG only requires introduction of short peptide tags to DNA aptamers and proteins, thus this method is not limited by certain combinations of aptamer and proteins of interest. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2013.05.025 ACKNOWLEDGMENTS
where a is the y value at the top plateau of the curve, b is the slope of the linear range in the curve, c is the thrombin concentration inducing the mean response between a and d, d is the y value for the blank (no analyte), x is the thrombin concentration, and y is the relative BAP activity. In the doseeresponse curve, BAP-aptamer conjugates yielded typical responses of the sandwich assay depending on the thrombin concentration. Both the BAP-aptamer and (BAP)n-aptamer yielded sufficient signals for thrombin concentrations greater than 1 nM and saturated at 25 nM of thrombin. Their thrombin dynamic ranges were 1e25 nM, indicating that there was no difference between single-labeled and multi-labeled aptamers in terms of their binding behavior to thrombin. The very similar dynamic range suggests that the differences of signal amplification are derived from the number of labeled enzymes, supporting the idea that multiple labeling by a two-step reaction was achieved. In terms of sensitivity, the detection limit (DL) was evaluated as the minimum detectable concentration of thrombin. The DL response was calculated as the mean of three blanks adding standard deviations (7,41). For the BAP-aptamer, the blank signal was 0.347 0.0273, leading to a DL of 0.61 nM. For the (BAP)n-aptamer, the blank signal was 0.782 0.0622, leading to a DL of 0.12 nM. A DL in the picomolar range is standard for this assay except for electrochemical detection, indicating that the aptamer is functioning sufficiently as a recognition element. Considering that the Kd is at the nanomolar level for the label-free aptamer, a narrow dynamic range was obtained from labeling, but higher sensitivities were obtained. Consequently, multi-labeling yielded a lower DL without altering the binding affinity of the aptamer to thrombin significantly. Finally, we tested BAP-TEO-aptamer conjugates with an aim to improve the detection results. The immobilization of functional
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Tailing DNA aptamers with a functional protein by two-step enzymatic reaction Mari Takahara,1 Kounosuke Hayashi,1, 3 Masahiro Goto,1, 2 and Noriho Kamiya1, 2, * Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan,1 Center for Future Chemistry, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan,2 and Hitachi Aloka Medical, Ltd., 6-22-1 Mure, Mitaka-shi, Tokyo, Japan3 Received 22 April 2013; accepted 15 May 2013 Available online 25 June 2013
An efficient, quantitative synthetic strategy for aptamer-enzyme conjugates was developed by using a two-step enzymatic reaction. Terminal deoxynucleotidyl transferase (TdT) was used to first incorporate a Z-Gln-Gly (QG) modified nucleotide which can act as a glutamine donor for a subsequent enzymatic reaction, to the 30 -OH of a DNA aptamer. Microbial transglutaminase (MTG) then catalyzed the cross-linking between the Z-QG modified aptamers and an enzyme tagged with an MTG-reactive lysine containing peptide. The use of a Z-QG modified dideoxynucleotide (Z-QG-ddUTP) or a deoxyuridine triphosphate (Z-QG-dUTP) in the TdT reaction enables the controlled introduction of a single or multiple MTG reactive residues. This leads to the preparation of enzyme-aptamer and (enzyme)n-aptamer conjugates with different detection limits of thrombin, a model analyte, in a sandwich enzyme-linked aptamer assay (ELAA). Since the combination of two enzymatic reactions yields high site-specificity and requires only short peptide substrates, the methodology should be useful for the labeling of DNA/RNA aptamers with proteins. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Enzyme-linked aptamer assay; DNA aptamer; DNA-enzyme conjugate; Microbial transglutaminase; Thrombin; Terminal deoxynucleotidyl transferase]
Nucleotide aptamers are nucleic acids that are able to bind target molecules with high affinity and specificity, which make them promising biorecognition elements (1,2). Compared with general receptor molecules such as antibodies, aptamers have several advantages as nucleic acids (3). Their in vitro production (SELEX; systematic evolution of ligands by exponential enrichment) (2) allows easy chemical synthesis, modification, and high stability after modification. These unique properties suggest their potential for applications in therapeutics, diagnosis, and analysis (3e6). Labeling of aptamers with functional molecules is important for practical applications. For signal amplification in molecular diagnostics (5), many DNA aptamer-enzyme conjugates have recently been developed using non-covalent interactions such as the biotineavidin interaction (7,8). Particular properties of DNA aptamers, such as high target specificity and affinity, render these molecules potential candidates for functionalization of nanomaterials (9). Thus, introducing target specificity of aptamers to nanomaterials can extend the application of aptamer-nanomaterial hybrid to intracellular analysis, cancer cell imaging and drug delivery. In order to maximize the functions of aptamer-enzyme/nanomaterial conjugates, site-specific labeling is required. For site-specific covalent linking of aptamer and enzyme, we herein propose a novel two-step tailing method using terminal deoxynucleotidyl
* Corresponding author at: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan. Tel.: þ81 92 802 2807; fax: þ81 92 802 2810. E-mail addresses: [email protected], nori_kamiya@ mail.cstm.kyus (N. Kamiya).
transferase (TdT) (10e13) and microbial transglutaminase (MTG) (14,15). The label was introduced by enzymatic reaction specifically to the 30 -OH termini of aptamers, enabling the preparation of bifunctional aptamer-enzyme conjugates. The aptamer tailing was limited by the ability of the DNA/ RNA polymerase to incorporate modified deoxynucleoside triphosphates (6). In addition, template-independent tailing is necessary because aptamers recognize target molecules in a single strand (1,2), but most polymerases requires both template nucleic acids and a primer (16). There is an unusual polymerase, TdT, which is able to catalyze template-independent addition of nucleotides to the 30 -OH termini of double- and single-stranded nucleic acids (16). TdT also incorporates modified nucleotides which leads to terminal labeling of certain molecules such as biotin and digoxigenin (17e22). However the sizes of enzyme-modified nucleotides are too large for TdT to recognize as substrates. Instead, we used MTGmediated cross-linking between modified aptamers and enzymes, and TdT for introducing MTG-reactive substrates to aptamers. MTG catalyzes a substrate specific acyletransfer reaction between the ε-amino group in lysine (Lys) and the g-carboxamide group in glutamine (Gln) (23). The use of MTG is crucial to maximize both the functions of aptamers and the labeled enzyme. DNA-protein conjugates (24,25) are usually synthesized by chemical modification and it depends on the functional groups of the target proteins. Random orientation of the reactive groups on the protein surface yields heterogeneous chemical conjugates with partial loss of function. By taking advantage of the high site-specificity of MTG, these problems are avoided, enabling attachment at the desired position (26). In our
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.05.025
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previous research, we synthesized an acyl-donor nucleotide substrate, 5-[3-(Z-Gln-Gly-amido)-1-(E)-propenyl]-20 -deoxyuridine 0 5 -triphosphate (Z-QG-dUTP), and incorporated it into DNA using PCR (27). In parallel, an acyl-acceptor Lys-containing tag (K-tag) sequence was fused to the N-terminus of the target protein, and site-specific labeling of Z-QG containing DNA using a K-tagged enzyme was achieved. In this study, we describe the use of a combination of TdT and MTG for site-specific tailing of a DNA aptamer with single or multiple enzymes. Thrombin was selected as a model analyte in order to validate the function of the enzyme-aptamer conjugates obtained. TdT incorporated Z-QG modified nucleotides into thrombin binding aptamers, and Z-QG labeled aptamers were conjugated with K-tagged bacterial alkaline phosphatase (BAP) using MTG (Fig. 1). The number of labeled BAPs can be controlled by using Z-QG-dUTP or Z-QG-ddUTP, a dideoxynucleotide analog, as terminators of the TdT reaction (18,19,28). The obtained BAP-aptamer conjugates were applied in a sandwich enzyme-linked aptamer assay (ELAA) (29e32) for thrombin to analyze both the functions of the aptamers and the labeled enzymes. In the sandwich ELAA, the 29-mer thrombin-binding aptamer (33) was immobilized as the primary aptamer, and the 15-mer thrombin-binding aptamer (34) modified by the two-step enzymatic reaction was the secondary aptamer. MATERIALS AND METHODS Chemicals Recombinant terminal deoxynucleotidyl transferase (TdT), and NBT/BCIP stock solution were purchased from Roche Applied Science (Basel, Switzerland). Z-QG-dUTP, Z-QG-ddUTP, 5-[3-(15-Z-Gln-Gly-amido-4,7,10,13-tetraoxapentadecanoylamido)-1-(E)-propenyl]-20 -deoxyuridine 50 -triphosphate (Z-QG-TEOdUTP), and 5-[3-(15-Z-Gln-Gly-amido-4,7,10,13-tetraoxapentadecanoylamido)-1-(E)-
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propenyl]-20 ,30 -deoxyuridine 50 -triphosphate (Z-QG-TEO-ddUTP) (Fig. S1) were synthesized at Gene Act, Inc. (Fukuoka, Japan). MTG was supplied by Ajinomoto Co., Ltd. (Tokyo, Japan) and was dissolved in 5 mM TriseHCl (pH 8.0). FX174 DNA-HaeIII Digest was purchased from New England Biolabs Ltd. (Ontario, Canada). Agarose S, albumin from bovine serum, Cohn fraction V, pH 7.0 (BSA), boric acid, magnesium chloride hexahydrate (99.9%), N-ethylmaleimide (NEM), sodium dodecyl sulfate (SDS) and tris (hydroxymethyl)aminomethane were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Formamide, potassium chloride, sodium chloride and urea were purchased from Kishida Chemical (Osaka, Japan). Tween20 was purchased from SigmaeAldrich (St. Louis, MO, USA). The Nunc Immobilizerstreptavidin plate (SA plate) was purchased from Nalge Nunc International Co. Ltd (Tokyo). The ECF substrate solution was purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK). Thrombin from human plasma was purchased from Merck Ltd. (Darmstadt, Germany). The thrombin binding aptamers (Tsukuba Oligo Service Co. Ltd., Tsukuba, Japan) used in this study have the following sequence: fluorescein isothiocyanate (FITC)emodified 15-mer aptamer with polyT15 tail, 50 -FITC-GGT TGG TGT GGT TGG TTT TTT TTT TTT TTT-30 ; biotinylated 29-mer aptamer with polyT20 spacer, 50 -biotin- TTT TTT TTT TTT TTT TTT TTA GTC CGT GGT AGG GCA GGT TGG GGT GAC T-30 . The underlined sequence, 50 - GGT TGG TGT GGT TGG-30 was reported by Bock et al. (34), and another sequence, 50 -AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-30 was reported by Tasset et al (33). These two thrombin binding aptamers have different binding sites for thrombin with Kds of 26 nM and 0.5 nM, respectively. Preparation of recombinant BAP A K-tagged BAP was prepared according to our previous reports. A K-tag sequence, MKHKGGGSGGGSGS (the underlined K is the MTG reactive residue) was fused to the N-terminus of BAP. The resultant BAP was abbreviated as NK14-BAP (35). The NK14-BAP gene was inserted into the pET22 (þ) plasmid vector. The recombinant NK14-BAP was produced using Escherichia coli BL21(DE3). The crude protein extract was purified using a HisTrap HP column 5 mL, and PD10-column (GE Healthcare) according to the manufacture’s instruction. The eluted protein was then purified using a size-exclusion chromatography (SEC) column equilibrated with 5 mM TriseHCl (pH 8.0). A SEC column was constructed by packing the XK16 column with Superdex 200 prep grade that were both purchased from GE Healthcare). All the chromatography experiments for the purification of NK14-BAP were conducted on a BioLogic DuoFlow Chromatography System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Purified NK14-BAP was analyzed by SDS-polyacrylamide gel electrophoresis (SDSPAGE) stained using Bio-Safe Coomassie (Bio-Rad Laboratories, Inc.) (Fig. S2). The
FIG. 1. Tailing aptamer with K-tagged BAP by a two-step enzymatic reaction. TdT incorporated Z-QG molecules into aptamers. MTG catalyzed cross-linking between Z-QG aptamers and K-tagged BAP (NK14-BAP). TdT needs only single strand DNA and nucleotides, which allows the use of MTG without purification of the primer.
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calculated molecular weight of NK14-BAP was 50 kDa. The protein concentration was determined with a Bicinchoninic Acid Protein Assay Kit (Thermo) using bovine serum albumin as a standard. Tailing aptamers with Z-QG modified nucleotides The Z-QG labeling reaction was conducted in a total volume of 100 mL by mixing the following compounds on ice: aptamer (FITC modified 15-mer), 5 mM; CoCl2, 5 mM; Z-QG-(TEO)ldUTP solution, 0.5 mM; or Z-QG-(TEO)l-ddUTP solution, 0.05 mM (l ¼ 0, 1); and 400 U/reaction TdT in reaction buffer (potassium cacodylate, 200 mM; TriseHCl, 25 mM; and BSA, 0.25 mg/mL, pH 6.6). The reaction solutions were incubated at 37 C for 2 h and terminated by heating at 94 C for 15 min. The TdT reaction products were directly subjected to denaturing polyacrylamide gel electrophoresis (PAGE) and analyzed by visualizing their fluorescently modified 50 -end. Denaturing PAGE A total of 1 mL TdT reaction products was diluted with ultra pure water to 5 mL then mixed with 5 mL formamide and 1 mL loading buffer. The denatured TdT reaction mixtures were subjected to electrophoresis using 15% denaturing gel containing 1 TBE buffer and 7 M urea at 280 V for 40 min. The gel was imaged with a fluorescent imager (Molecular Imager FX Pro, Bio-Rad Laboratories, Inc.) with an excitation wavelength of 488 nm and a 530 (15) nm band pass filter. The reaction products were visualized by their 50 -FITC (excitation wavelength of 488 nm and emission wavelength of 530 nm). Labeling Z-QG aptamers with NK14-BAP The TdT reaction solutions were subjected to a Probe Quant G50 spin column (GE Healthcare) to remove nonextended aptamers and unreacted Z-QG-(TEO)l-dUTPs or Z-QG-(TEO)l-ddUTPs. After purification, Z-QG aptamers were labeled with NK14-BAP using MTG. The MTG reaction mixture (300 mL) consisted of Z-QG aptamer (0.5 mM), NK14-BAP (2.5 mM; (ZQG-(TEO)l)m-aptamer, 1.25 mM; Z-QG-(TEO)l-aptamer), and MTG (0.1 U/mL) in 20 mM TriseHCl buffer (pH 7.4). The MTG reaction solutions were incubated at 4 C for 3 h and terminated by NEM (1 mM). The obtained BAP-aptamer conjugates were analyzed using 2.0% agarose gel electrophoresis in 1 TBE buffer. Electrophoresis was conducted at 135 V for 27 min. The gel was imaged using a fluorescent imager for aptamers as described previously. Ultimately, the gel was stained in NBT/BCIP solution following an established protocol below. NBT/BCIP coloring reaction In the coloredevelopment reaction, NK14-BAPcatalyzed dephosphorylation of 5-bromo-4-chloro-3-indolyl phosphate (BCIP), thereby priming the agarose gel for the blue precipitates to appear on the agarose gel following reduction of p-nitroblue tetrazolium chloride (NBT). NBT/BCIP stock solution (18.75 mg/mL NBT and 9.4 mg/mL BCIP) was dissolved in NTMT buffer (100 mM NaCl, 100 mM TriseHCl, 50 mM MgCl2, pH 9.5). The gel was incubated in prepared NBT/BCIP solution at 37 C for 40 min. General enzyme-linked aptamer assay (ELAA) protocol This protocol is based on our previous research. All chemicals were diluted with Tris Buffered Saline (TBS; 25 mM TriseHCl, 137 mM NaCl, 2.68 mM KCl, pH 7.4), unless specified. All the steps were carried out at 37 C. The SA plate was washed three times with TBST (TBS containing 0.05% v/v Tween20) after the addition of each biomolecule. Before the immobilization, the biotinylated 29-mer aptamer was heated at 94 C for 15 min to anneal the DNA strand and then cooled on ice for 10 min in order to keep the DNA structure unfolded. The biotinylated 29-mer aptamer (200 nM, 100 mL) was immobilized on the SA plate. After incubation for 1 h and washing, thrombin (100 nM, 100 mL) was added to the SA plates. After incubation for 1 h and washing, the BAP-aptamer conjugate (50 nM, 100 mL) was added to the SA plate. After incubation for 1 h and washing, ECF substrate (1 mM, 100 mL) was added to the SA plate. The ECF substrate was diluted with NTMT buffer. After incubation for 30 min, the enzymatic product was detected by an LS-55 Fluorescence Spectrometer (PerkinElmer) using the fluorescence intensity at 560 nm with an excitation wavelength of 430 nm.
RESULTS AND DISCUSSION Tailing aptamers with Z-QG modified nucleotides TdT adds a nucleotide or nucleotide analog to the 30 -OH end of singlestranded DNA. To introduce Z-QG molecules, Z-QG-ddUTP and ZQG-dUTP were employed in a TdT-catalyzed tailing reaction. The dideoxynucleotide analogue was prepared for the single Z-QG labeled aptamer because TdT initiates homopolymerization by recognizing the 30 -OH end of nucleic acids. We also focused on the effect of polyethylene oxide (PEO) linker on the TdT reaction by introducing Z-QG-TEO-ddUTP and Z-QG-TEO-dUTP. The model aptamer was a thrombin-binding 15-mer aptamer, and a 50 -FITC modified 15-mer aptamer with polyT15 tail was designed for the TdT reaction (36). Fig. 2 shows the result of denaturing PAGE analysis of the TdT reaction products. Lanes 2e5 (TdT reaction products) showed the uniform mobility and the disappearance of the original aptamer band (lane 1). This result suggests all aptamers were labeled with
FIG. 2. Denaturing PAGE analysis of the TdT reaction products. In the TdT reaction mixture, no nucleotides (lane 1), Z-QG-ddUTP (lane 2), Z-QG-TEO-ddUTP (lane 3), ZQG-dUTP (lane 4), and ZQG-TEO-dUTP (lane 5) were incubated with aptamers and CoCl2. M: FX174 DNA-HaeIII digest treated with FITC-ddUTP, CoCl2, and TdT.
the Z-QG moiety and TdT can recognize all the Z-QG modified ddUTP/dUTPs as substrates. Namely, perfect conversion of aptamers was achieved in the TdT reaction regardless of Z-QG and PEO modifications. Z-QG-dUTP labeled aptamers were shifted to higher mobility than Z-QG-ddUTP labeled ones because of the addition of several Z-QG molecules (lanes 2, 3 and lanes 4, 5, respectively). The average number of labeled Z-QG was estimated by the absorbance of 296 nm (27). Incubation with the dideoxynucleotide analogs (ZQG-ddUTP or Z-QG-TEO-ddUTP) yielded 1:1 stoichiometric labeling of the aptamer with single Z-QG moiety. On the other hand, the number of Z-QG moiety per aptamer was calculated to be approximately two for Z-QG-dUTP and three for Z-QG-TEO-dUTP, respectively. The resultant Z-QG multi-labeled aptamers (i.e., (ZQG)m-aptamer and (Z-QG-TEO)m-aptamer) showed a slower mobility shift than Z-QG single-labeled aptamers. The PEO linker effect is shown in lane 5. TdT polymerizes nucleotides randomly therefore the reaction product is characterized by various tail lengths and appears as a broad band in Fig. 2. (ZQG)m-aptamers were a broad band because of random addition (Fig. 2, lane 4), but (Z-QG-TEO)m-aptamers resulted in narrower size distribution (Fig. 2, lane 5), associated with the PEO linker effect. The efficiency of the TdT reaction depends on the type of nucleotides that affect the structure of the 30 -termini of DNA/RNA. The effect of the PEO linker is not yet fully understood, but multiple labeling of Z-QG might increase the hydrophobicity of the 30 termini of the aptamer resulting in an inability of TdT to access the sterically hindered 30 -OH end. The introduction of the PEO linker may reduce this assumed effect and thus allow a more controlled mean tail length. Labeling Z-QG aptamers with NK14-BAP Transglutaminase catalyzes the acyl transfer reaction between the ε-amino group in Lys and the g-carboxamide group of Gln in peptides and proteins (37,38). Microbial transglutaminase from Streptomyces mobaraensis (MTG) can catalyze conjugation of a K-tagged enzyme and Z-QG modified DNA in a Ca2þ independent manner (14). Z-QG is a neutral dipeptide substrate recognized by MTG as an acyl-donor. A recombinant protein, K-tagged BAP (NK14-BAP), has an acylacceptor tag sequence (K-tag) at the N-terminus (35). We examined the site-specific labeling of Z-QG aptamers with NK14BAP using MTG. Fig. 3 shows the agarose gel electrophoresis analysis of the obtained BAP-aptamer conjugates. Each lane shows the mobility shifts and the disappearance of the original Z-QG aptamer bands. Since few residual Z-QG aptamers were observed, almost all Z-QG labeled aptamers were converted to NK14-BAP-aptamer conjugates in the MTG catalyzed reaction (Fig. 3a). As shown in Fig. 3b, NK14-BAP was stained by the BAP-catalyzed NBT/BCIP coloring reaction. The mobility shifts of NK14-BAP
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FIG. 3. Agarose gel electrophoresis analysis of MTG reaction products. (a) DNA aptamers were imaged with FITC, and (b) NK14-BAP was detected by the BAP-catalyzed NBT/BCIP colouring reaction in the same gel. Lane 1: aptamer alone. A mixture of NK14-BAP and Z-QG-aptamer (lanes 2 and 4), Z-QG-TEO-aptamer (lanes 3 and 5), (Z-QG)m-aptamer (lanes 6 and 8) or (Z-QG-TEO)m-aptamer (lanes 7 and 9) was incubated without MTG (lanes 2, 3, 6, and 7) or with MTG (lanes 4, 5, 8, and 9). M: FX174 DNA-HaeIII Digest.
corresponded to that of the aptamers. This observation supports the mobility shift derived from labeled NK14-BAP. Conjugated BAP showed higher mobility than unconjugated BAP because of the negative charge of aptamers. The different mobility in electrophoresis could be determined by their charge differences. Similar results were reported in the case of a thrombin-aptamer complex (31). The Z-QG multi-labeled aptamers were shifted to lower mobility compared to the Z-QG single-labeled aptamers because of multiple labeling of NK14-BAP. In addition, MTG can catalyze efficient cross-linking in the presence of PEO linker (Fig. 3b, lanes 5 and 9). From quantitation of incorporated Z-QG, multiple labeled aptamer would have at most 2- or 3-labeled BAPs, resulting in high BAP activity in NBT/BCIP coloring reaction (Fig. 3b, lanes 8 and 9). In contrast, low BAP activity of single-labeled aptamer was observed (Fig. 3b, lanes 4 and 5). The lower activity was resulting from the difference of conjugated number of BAP. By the two-step enzymatic reaction, preparation of a single-enzyme labeled aptamer (i.e., BAPaptamer) and a multiple-enzyme labeled aptamer (i.e., (BAP)naptamer) was successfully conducted. The above described enzymatic strategy should prove useful for the development of various aptamer-protein conjugates because the combination is not limited in this system and quantitative conversion is achieved. MTG requires only short reactive peptides containing Gln in the aptamer and Lys in the recombinant protein. The Z-QG is easily introduced into the aptamer in the TdT reaction using Z-QG modified nucleotides. ELAA with BAP-aptamer conjugates BAP-aptamer conjugates labeled by a two-step enzymatic reaction were then applied to the sandwich ELAA for thrombin (Fig. 4a). The sandwich assay was performed using streptavidin-coated 96-well microplates (SA plates). The biotinylated 29-mer aptamer (200 nM) was immobilized on the SA plates as a primary aptamer, and thrombin (100 nM) was added. After thrombin capture, a BAPaptamer conjugate (50 nM) employed as a secondary aptamer was added for detection. The SA plates were washed between each step to remove residual compounds. The conjugate concentrations were optimized in accordance with the response of BAP-aptamer (Fig. S3). Both functions of aptamer and BAP were evaluated by measuring the relative activity of BAP. First, we investigated the nonspecific reaction of the BAPaptamer conjugates. The biotin-streptavidin system, one of the most general (non-covalent) tailing methods, is broadly used because of its natural high affinity. However this strong interaction also causes a high level of nonspecific reaction leading to falsepositive results. Since the BAP-aptamer conjugates in this study were covalently tailed, the nonspecific reaction could be minimized. As shown in Fig. 4b, in the presence of all components
(Fig. 4b, lane 5), the (BAP)n-aptamer showed increased relative activity although a very low BAP activity was observed in the absence of each component (Fig. 4b, lanes 1e3). The BAP-aptamer showed lower activity compared with the (BAP)n-aptamer. The decreased activity from the single labeling made it difficult to distinguish between specific and nonspecific binding. We also examined the selectivity of BAP-aptamer conjugates by mixing other protein (BSA) instead of thrombin. Little activity was observed as shown in Fig. 4b, lane 4. These results confirmed that BAP-aptamer conjugates selectively bind to thrombin without nonspecific adsorption especially for the (BAP)n-aptamer. In the presence of thrombin, a 4-fold increase in activity of (BAP)naptamers to BAP-aptamers was observed. This difference should result from the number of labeled BAP or the effect of labeled BAP on binding affinity. To study the dynamic range and sensitivity of the assay using labeled aptamers, BAP-aptamer conjugates were incubated with thrombin at various concentrations in the range of 0e100 nM. A doseeresponse curve for thrombin was carried out (Fig. 5). The curve was fitted by sigmoidal regression using the following four-
FIG. 4. ELAA applications. (a) Scheme of the sandwich ELAA. (b) Relative BAP activities of BAP-aptamer conjugates under various conditions. The immobilized aptamer on the plate captured thrombin, then the BAP-aptamer conjugate bound to thrombin (lane 5). As a negative control, ELAA was performed in the absence of BAP-aptamer conjugate (lane 1), biotinylated aptamer (lane 2) and thrombin (lane 3). Instead of thrombin, BSA was utilized (lane 4). The BAP activities were relative to the value obtained in the presence of biotinylated aptamer, thrombin and BAP-aptamer (lane 5, white histogram). All error bars represent standard deviations obtained through the detection of three parallel samples. Open bars, BAP-aptamer; closed bars, (BAP)n-aptamer. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons within all lanes. *p < 0.05 versus lanes 1e4 for each conjugate.
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J. BIOSCI. BIOENG., biomolecules on a solid surface was shown to decrease their functions as a result of their limited movement (42). The PEO linker provides labeled enzymes flexibility, which could increase enzymatic activity at a solid surface. The dynamic range of the BAPTEO-aptamer was 1e25 nM and its DL was 0.36 nM. Since an improved result was obtained by using the BAP-TEO-aptamer, we also investigated the (BAP-TEO)n-aptamer. The dynamic range of the (BAP-TEO)n-aptamer was 1e100 nM and its DL was 0.65 nM (Fig. S4). The introduction of the PEO linker to the BAP multilabeled aptamer resulted in a wider dynamic range but an improvement in the DL was not observed.
FIG. 5. Doseeresponse curve for thrombin. ELAA was performed under different concentrations of thrombin in the range of 0e100 nM. The BAP activities were relative to the value obtained in the presence of thrombin (100 nM) and BAP-aptamer. All error bars represent standard deviations obtained through the detection of three parallel samples. Open circles, BAP-aptamer; open triangles, BAPeTEO-aptamer; closed circles, (BAP)n-aptamer.
parameter logistic equation with KaleidaGraph (Synergy Software, Reading, PA, USA) (7,39,40): y ¼ dþ
ad 1 þ ðx=cÞb
(1)
Conclusions Tailing aptamers with K-tagged enzymes by a two-step reaction was achieved. The two-step reaction was useful to modify all aptamers, providing 100% conversion. Because of its template-independency and its activity toward a wide range of substrates, TdT is an efficient polymerase for tailing DNA aptamers. MTG can catalyze site-specific cross-linking between Z-QG modified aptamers and K-tagged proteins which avoids inactivation of both functions. The functions of both aptamers and enzymes were confirmed using ELAA analysis. TdT can incorporate mono or several modified nucleotides to the 30 -OH end of nucleic acids. This is a controlled single- or multi-labeling reaction compared to random chemical conjugation. Even in the presence of PEO modification, TdT and MTG can recognize the aptamers as substrates, and the two-step reaction proceeds efficiently. The two-step reaction catalyzed by TdT and MTG only requires introduction of short peptide tags to DNA aptamers and proteins, thus this method is not limited by certain combinations of aptamer and proteins of interest. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2013.05.025 ACKNOWLEDGMENTS
where a is the y value at the top plateau of the curve, b is the slope of the linear range in the curve, c is the thrombin concentration inducing the mean response between a and d, d is the y value for the blank (no analyte), x is the thrombin concentration, and y is the relative BAP activity. In the doseeresponse curve, BAP-aptamer conjugates yielded typical responses of the sandwich assay depending on the thrombin concentration. Both the BAP-aptamer and (BAP)n-aptamer yielded sufficient signals for thrombin concentrations greater than 1 nM and saturated at 25 nM of thrombin. Their thrombin dynamic ranges were 1e25 nM, indicating that there was no difference between single-labeled and multi-labeled aptamers in terms of their binding behavior to thrombin. The very similar dynamic range suggests that the differences of signal amplification are derived from the number of labeled enzymes, supporting the idea that multiple labeling by a two-step reaction was achieved. In terms of sensitivity, the detection limit (DL) was evaluated as the minimum detectable concentration of thrombin. The DL response was calculated as the mean of three blanks adding standard deviations (7,41). For the BAP-aptamer, the blank signal was 0.347 0.0273, leading to a DL of 0.61 nM. For the (BAP)n-aptamer, the blank signal was 0.782 0.0622, leading to a DL of 0.12 nM. A DL in the picomolar range is standard for this assay except for electrochemical detection, indicating that the aptamer is functioning sufficiently as a recognition element. Considering that the Kd is at the nanomolar level for the label-free aptamer, a narrow dynamic range was obtained from labeling, but higher sensitivities were obtained. Consequently, multi-labeling yielded a lower DL without altering the binding affinity of the aptamer to thrombin significantly. Finally, we tested BAP-TEO-aptamer conjugates with an aim to improve the detection results. The immobilization of functional
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