A Sol-Gel–Integrated Protein Array System for Affinity Analysis of Aptamer–Target Protein Interaction

Introduction

Aptamers are oligonucleotides bound to target molecules, such as proteins, small molecules, and chemicals. Aptamers are generally isolated from a synthesized random library (typically 10¹⁵ molecules) using an in vitro selection method, called SELEX (systematic evolution of ligands by exponential enrichment) (Gold et al., 1997; Zhao et al., 2006; Lee et al., 2007; Sevilimedu et al., 2008). SELEX typically requires more than 10 selection rounds and the intermediate rounds contain a decreasing number of unique sequences by repeating, partitioning, and amplifying, and the average affinity of each round will increase by the repeated selection (Hamaguchi et al., 2001; Shi et al., 2007). After selection, the selected individual aptamers with affinity to the target molecules should be evaluated for further applications, such as a lead material of drug discovery or development of sensing tools. The electric mobility shift assay (EMSA) is a typical method for measuring an aptamer–protein interaction, which can be separated from the free aptamer on the gel, and the total radioactivity on the gels can be applied to determine the specific activity of the aptamer (Fan et al., 2004; Park et al., 2009). Nevertheless, there are safety and handling concerns regarding radiolabeling methods. Therefore, nonradiolabeling methods, such as real-time analysis, have been developed.

To develop simplified and direct affinity measurement methods, others have recently reported the dissociation constant measurement of aptamers by surface plasmon resonance (Wang et al., 2007) and kinetic capillary electrophoresis (Yunusov et al., 2009) analysis. Although both methods can monitor the aptamer–protein binding affinity in real-time with a simplified and direct procedure, the low throughput of these methods is a considerable disadvantage for screening large number of candidate aptamers.

Previously, a sol-gel–based protein array was developed for protein–protein or antigen–antibody interaction assays. A comparison with the 2-dimensional microarray system showed that sol-gel can immobilize proteins with a broad range up to milligram level with high activity (Kim et al., 2006; Kwon et al., 2008). Therefore, the sol-gel–integrated protein microarray appears to be ideal for measuring affinity, because sol-gel can house proteins with different concentration ranges. In addition, the same protein arrays produced can be used for high-throughput measurements of a large number of aptamers screened.

In this study, this sol-gel material was used to immobilize the target protein, TATA-binding protein (TBP), and RNA aptamers (TBPapt 12, 15, 16, 18, 24, and 26) that were selected from a previous study (Kim et al., 2006; Lee et al., 2007; Park et al., 2009). As the 3-dimensional nanoporous structure of the sol-gel droplets can allow rapid diffusion, the introduced aptamers can interact with the immobilized TBPs within a few seconds, because labeled aptamers travel along the network of channels (Kim et al., 2006; Lee et al., 2007; Park et al., 2009). Therefore, it is expected that the binding affinity can be deduced from the aptamer–protein interaction results under conditions in which different amounts of protein are immobilized in the microarray (see Supplementary Fig. S1; Supplementary Data are available online at www.liebertonline.com/nat). This system allows quantitative binding measurements of the aptamer–target interactions in a high-throughput manner and can be potentially applied to automatic processes as a part of the SELEX lab-on-a-chip system.

Materials and Methods

Microarray preparation

Sol-gel materials were used to immobilize the proteins according to the manufacturer's recommendations (SolB complete kit; PCL, Inc.; www.pclchip.com). In detail, using the sol-gel formulation (SolB I 25%, SolB II 7.5%, SolB III 5%, SolB H 12.5%, and SolB S 12.5%), the 5 different concentrations of TBP described in Fig. 1 were mixed with the SolB reagents and arrayed with the controls (R: reference control with Cy-3 dUTP) using a noncontact microdispensing instrument [SolB Desktop Arrayer (PCL, Inc.); sciFLEXARRAYER (Scienion)] under high-humidity conditions (∼60%, 20°C). Arraying was performed in 8-mm-diameter wells of a 96-well plate and cured for 16 hours to allow gelation according to the manufacturer's recommendation. The final arrayed sol-gel microdroplet volume was calculated using autodrop volume detection software (Scienion). The morphology of the sol-gel spot was observed by optical microscopy (KH-770; HIROX Company).

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FIG. 1.

(A) In 1 well of a microplate, 10 duplicate sol-gel microdroplets with different protein concentrations [from 0 to 800 nM (0.02 mg/mL of TBP)] were spotted along with 2 reference controls (R: reference control with Cy-3 dUTP). The left panel shows the microdroplets' position with different concentrations of TBPs. The right panel is the scanned image after affinity measurement assay of a spotted well between TBPs and a labeled aptamer. (B) Each single sol-gel microdroplet has a distinct 3-dimensional morphology (the average volume of 1 droplet was ∼50 nL). The spot diameter and height are ∼250 and 20 μm, respectively. The morphology was imaged using an optical microscope (KH-7700). TBP, TATA-binding protein.

Results and Discussion

For the aptamer binding affinity test, 5 different TBP concentrations (from 0 to 800 nM) were used to measure the K_d of the aptamers. The TBP-containing sol-gel mixture was prepared and 8 duplicate TBP-containing sol-gels were arrayed along with the 2 negative controls (without protein) and 2 reference controls (R; reference positioning, with Cy-3–labeled antibody) within the 8-mm-diameter wells of 96-well microtiter plates using a noncontact piezoelectric liquid dispenser, as described in the Materials and Methods section (Fig. 1A). The 3-dimensional nanostructured composite prepared by the sol-gel process provides an aqueous environment that preserves the biological activity of immobilized proteins and might enhance the biomolecule stability (Frenkel-Mullerad and Avnir, 2005; Kim et al., 2006). Figure 1B shows an optical microscopy image of the sol-gel microdroplet. Upon entrapment, biomolecules are encased by the hydrated silica in the pores tailored to the size of the embedded target biomolecules. Moreover, the interacting partner has access through the sol-gel network to bind with its target specifically.

The selected aptamers (TBPapt 12, 15, 16, 18, 24, and 26; Table 1) were transcribed and labeled at the C-terminus with Cy-3 dUTP as described in the Materials and Methods section. Each Cy-3–labeled aptamer in the binding buffer was added to the individual TBP arrayed well after soaking and blocking of the wells and incubated for 1 hour at room temperature. After washing 3 times with a washing buffer, the resulting plate chip well was scanned and analyzed using a 96-well fluorescence scanner and the appropriate software program, as described in the Materials and Methods section (Fig. 1A, binding assay feature). The background intensity was subtracted from the signal intensity of each microdroplet.

The dissociation constants (K_d) were calculated by plotting the fluorescent intensity of the bound aptamers as a function of the TBP concentration, and the specific binding data were then fitted to the following nonlinear regression equation (Xu et al., 1996) using Sigmaplot 10.0 software: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} y = ( B_{\max} \, \bullet \, [ { \rm yTBP} ] ) / (K_{ \rm d} \, + \, [ { \rm yTBP} ] ) \end{align*} \end{document}

where y is the total binding (fluorescent intensity in sol-gel), B_max is the maximum fluorescent binding activity, and K_d is the dissociation constant.

As shown in Fig. 2 and Table 1, the dissociation constants (K_d) of all aptamers were in the nanomolar range, with TBPapt 12 showing the highest affinity (K_d ∼2.7 nM). In the case of TBPapt 16, the binding affinity previously measured by EMSA ranged from 3 to 10 nM (Fan et al., 2004), and the sol-gel–integrated assay showed a binding affinity of ∼8 nM K_d (Fig. 2A). This suggests that the sol-gel–based binding affinity measurements fit well with conventional binding affinity measurements, suggesting their possible use as an alternative to the conventional method.

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FIG. 2.

Binding affinity of the aptamers to TBP. (A) Sol-gel chip-based binding affinity of TBPapt 16 was compared with the conventional method, EMSA. Left: Measured K_d by sol-gel–integrated protein chip assay (protein concentrations: 0, 10, 100, 200, and 400 nM). Right: Binding affinity previously measured by EMSA (Fan et al., 2004). (B) Individual binding affinities of the aptamers (TBPapt 12, 15, 16, 18, 24, and 26) to TBPs were measured using the sol-gel chip assay. EMSA, electric mobility shift assay.

Aptamer candidates isolated from SELEX show affinity and specificity over a wide range against the applied target. The target binding affinity and functional activity of individual members should be characterized and aptamers with the highest affinity can be used as capturing ligands in molecular imaging, drug discovery, or biosensor studies. The sol-gel–integrated protein chip system in the present study allows easy measurement of the binding affinities of several tens of aptamer candidates isolated using the SELEX process simultaneously. The approach described herein can be also used to set up an automatic workstation because of the mechanical operability from the start (sol-gel spotting) to finish (affinity analysis) of the overall procedure. Therefore, a highly integrated automatic system can be extended further to realize high-throughput aptamer affinity analysis against a large number of proteins simultaneously.