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Use of aptamers in immunoassays
Molecular Immunology 70 (2016) 149–154
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Review
Use of aptamers in immunoassays Roald Nezlin ∗ Department of Immunology, Weizmann Institute of Science, Rehovot 7610001, Israel
a r t i c l e
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Article history: Received 21 October 2015 Received in revised form 2 December 2015 Accepted 15 December 2015 Keywords: Aptamers Apta-sorbents Ig detection Aptamers instead of antibodies
a b s t r a c t Aptamers, short single-chain DNA or RNA oligonucleotides, react specifically with small molecules, as well as with proteins. Unlike antibodies, they may be obtained relatively easily. Aptamers are now widely employed in immunological studies and could replace antibodies in immunoassays. In this short review, methods for immobilizing aptamers on various insoluble materials (so-called apta-sorbents) are described. Recent findings on their use in the detection and isolation of immunoglobulins and their application in various immunoassays are also discussed. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction For a long time immunological methods have been applied in various areas of biological and clinical research. Serum proteins, antibodies, form integral parts in all variants of immunoassays. These large protein molecules are highly specific and react with strong affinity with broad array of ligands. However, the production of antibodies is a costly, time-consuming process. This especially holds true for monoclonal antibodies. Furthermore, antibodies easily lose their specific activity after denaturation; therefore require mild conditions during experimentation. Aptamers are short, synthetic single chain DNA or RNA oligonucleotides which fold into unique three-dimensional structures. They react specifically, and with high affinity, with various ligands, low-molecular weight molecules as well as with proteins. Methods to prepare aptamers in 1990 by means of SELEX procedure were described (Tuerk and Gold, 1990; Ellington and Szostak, 1990). During the subsequent 25 years, aptamers have been widely used in various areas of biological research, including immunological studies (Nezlin, 2014), and intensively applied in the development of many effective analytical methods (Mairal et al., 2008; Iliuk et al., 2011; Li et al., 2014). The importance of aptamers in a wide range of applications of is based on several factors, among them the following: the production of aptamers is relatively cheap; aptamers are stable products and after denaturation aptamer molecules can be rapidly regenerated, with no loss of specificity; aptamers can
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be modified, particularly by conjugation with various functional groups such as fluorophores or biotin, without loss of their specific activity. This review summarizes the ways in which aptamers can be applied in various methodologies commonly used in immunological research. Immobilization of aptamers on insoluble carriers is examined, followed by descriptions of the aptamer application aimed at the detection and isolation of immunoglobulins. Furthermore, some widely applied techniques that entail the application of aptamers are discussed. 2. Immobilization of aptamers on insoluble carriers (apta-sorbents) Following the advent of aptamers, many means to immobilize them were suggested. Various insoluble supports were utilized, among them gold nanoparticles (Reinemann and Strehlitz, 2014; Sassolas et al., 2011), porous silicon (Urmann et al., 2015), glass (Huy et al., 2011) and magnetic beads (Centi et al., 2008; Kökpinar et al., 2011; Deng et al., 2014; Zhu and Walter, 2015; Ozalp et al., 2015), carbon and silica nanoparticles (Lin et al., 2014; Jo et al., 2015), and graphene (Ping et al., 2015) among others (Table 1). Two types of aptamer immobilization—non-covalent or covalent—have been used. The first variant is the simpler of the two, is based on the ability of aptamers to adsorb on the surface of a solid phase. The negative charges of sugar–phosphate backbone facilitate aptamer adsorption (Li et al., 2014). Modifications of aptamers as well as solid surfaces could be correspondingly performed. Covalent coupling is preferable as stable immobilization is important in many assays, among them chromatographic
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Table 1 Some insoluble carriers used for aptamer immobilization. Gold nanoparticles Silica nanoparticles Carbon nanoparticles Magnetic beads Glass beads Glass slides Graphene Porous silicon Tresyl resin Activated Sepharose Oxidized cellulose particles Oxidized paper
techniques. Physically adsorbed aptamer molecules are linked to a solid surface in random fashion; hence, not all of them are accessible to ligand. Covalently immobilized aptamers are arranged on solid matrix at optimal orientations. For covalent immobilization terminal nucleotides are modified and coupled with an appropriated solid surface. Such an approach was utilized for example, to immobilize of cyanuric-activated aptamers on amino-modified glass slides (Walter et al., 2008). The three-dimensional structure of aptamers is more likely to fold correctly to enable their coupling with a target, if a linker is introduced as a spacer between aptamer molecules and the surface. Sequence of thymidine nucleotides or polyethylenglycol is often used as linkers for immobilization of aptamers on gold or magnetic beads (Balamurugan et al., 2008a; Zhu and Walter, 2015). During the past decade immobilization of aptamers on gold plates or on gold nanoparticles was applied in many studies. For coupling with gold, thiol-alkane group is linked to an oligonucleotide chain terminal. This simple procedure involves arrangement of an aptamer monolayer in an orderly fashion. The resulting apta-sorbents are stable. Following addition of target molecules aggregated gold nanoparticles with immobilized aptamers change color from red to pinkish/purple and the aptamer–target interaction can be registered by the naked eye with no need for any special equipment (Elghanian et al., 1997; Storhoff et al., 1998). Surfaces such as glass slides or particles, magnetic beads and gold with acidic groups are useful platforms for the immobilization of modified aptamers. For covalent linkage several chemical groups on surfaces are used, among them are hydroxyl, amine, and carboxylic acid groups which could be further modified. An example of such procedure is the immobilization of aptamers activated with cyanuric chloride on amino-modified glass slides (Walter et al., 2008). Fine cellulose particles (Gurvich and Lechtzind, 1982) or paper (Nezlin, 2005) oxidized by NaIO4 to introduce aldehyde groups could be applied to immobilize aptamers covalently through their amino-terminal groups. A suspension of fine cellulose particles can be stored in water at 4◦ for prolonged time. Pieces of oxidized paper can be employed in strip tests. As aptamers are small molecules, their density on solid surfaces is significantly higher than that of immobilized protein molecules. On a given solid matrix it is possible to fix aptamers with different specificities simultaneously; for example, directed at two different epitopes of a single protein molecule or at two separate molecules. In some assays, such types of interactions can achieve higher levels of sensitivity (Deng et al., 2014). Dual aptamer-silica nanoparticles have been applied as effective diagnostic probes in instances of breast cancer (Jo et al., 2015). One aptamer recognized the mucin 1 cell line and a second, the epidermal growth factor cell line. Such a dual aptamer system was more effective than single aptamer particles for diagnosis of breast cancer. Moreover, the sensitivity of
the diagnostic probe was very high with a detection limit of one cell/100 l. Apta-sorbents have been successfully employed in various methodologies, including immunoassays, as well as in many techniques for the detection and extraction of various analytes in affinity chromatography, affinity capillary electrophoresis, and electrochromatography, among others (Romig et al., 1999; Giovannoli et al., 2008; Balamurugan et al., 2008b; Pichon et al., 2015). 3. Detection and isolation of immunoglobulins Over the past decade several aptamers specific to immunoglobulins (Ig) of human, mouse and rabbit origin were obtained. Interest in this problem is quite understandable, as Ig-specific aptamers after immobilization could be applied not only to detect Igs but also to the industrial-scale isolation of therapeutic antibodies (Ramsland et al., 2015). 3.1. Immunoglobulin G A 23-long RNA aptamer, Apt8-2, with the ability to interact specifically and with high affinity with the Fc region of human IgG was prepared. IgGs of animal species do not react with this aptamer. For the aptamer’s reaction with Fc␥, the GGUGCU bulging motif was found to be indispensable (Miyakawa et al., 2008). The specific aptamer reactive structure is supported by the presence of the divalent ions (Ca2+ and Mg2+ ), without which no reaction with IgG was observed. The structure of the RNA aptamer in complex with the human IgG1 molecule was studied by X-ray crystallography at 2.15 A´˚ resolution (Sugiyama et al., 2008; Miyakawa et al., 2008; Nomura et al., 2010). The RNA aptamer combines with the Fc part of IgG1 at the same site as bacterial protein A; both reagents compete with each other for the reaction with Fc␥. The Fc␥ cell receptor binding site does not overlap with the aptamer and protein A sites. Several non-electrostatic forces, such as hydrogen bonds and van der Waals forces, were shown to be involved in the aptamer–Fc interaction. Three sites on the surface of human Fc␥ are important for its interaction with the aptamer. They include amino-acid residues 316–324; 338–344; and 397–406. Among them residues 315, 342 and 404 are specific for human Fc␥. The spacial structure of human Fc␥ recognition sites is also differs from corresponding regions of animal IgGs. For preparation of apta-sorbents, the RNA aptamers were modified by attaching amino groups, via a linker to 3 or 5 ends (Miyakawa et al., 2008). The modified aptamers were immobilized on Tresyl resin or NHS-activated Sepharose. These adsorbents effectively bound IgG from human serum, as well as therapeutic chimeric or humanized antibodies from cell culture medium. Bound proteins were eluted by neutral buffer containing 10 mM EDTA. This chelating agent changes the aptamer structure by removing divalent ions and disrupts aptamer–IgG complex. The apta-sorbent could be regenerated many times by means of 6 M urea without loss of adsorbent capacity. Furthermore, preparation of the apta-sorbent is cheaper than that of the protein A-sorbent, an advantage most important for large-scale production of therapeutical antibodies. Aptamers specific to rabbit and mouse IgG were also obtained. The RNA aptamer R 18 specifically bound to native rabbit IgG but not to SDS-denatured rabbit IgG or to IgG of mouse or goat (Yoshida et al., 2008). R 18 also combined with isolated rabbit Fc␥; however the binding was significantly less effective than that with an intact IgG molecule. Two stretches of R 18, each three nucleotides long, are
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critical to its binding to the IgG molecule. It has been proposed that nearly the entire R 18 molecule combines with IgG. Mg2+ ions are required for the effective reaction of R 18 with IgG. Removal of these ions with EDTA resulted in dissociation of the R 18–IgG complex after which the aptamer could be again effectively used for purification of rabbit antibodies. The biotinylated R 18 was successfully used in blotting experiments as a secondary anti-rabbit IgG reagent. DNA and RNA aptamers that specifically reacted with mouse Fc␥ were prepared. The RNA aptamer MIG1 was found to specifically interact with the Fc region of three mouse IgG subclasses—IgG1, IgG2a and IgG3 (Sakai et al., 2008). Denatured IgG molecules, on the other hand, were unable to combine with the aptamer. IgG of four other species did not react with MIG-1, with the exception of rat IgG which cross-reacted with mouse IgG due to similarities in their amino-acid sequences. MIG-1 effectively recognized mouse IgG in blotting analysis. DNA aptamers that reacted with mouse IgG1 and IgG2a were isolated, using a modified SELEX procedure (Ma et al., 2013). These aptamers display a similar stem-loop secondary structure which is important for binding. Dot blot and electrophoretic mobility shift (EMSA) assays were utilized to study the binding activity of the aptamers. 3.2. Immunoglobulin M A DNA aptamer TD05 which specifically recognizes an epitope expressed on B lymphocytes and Burkitt’s lymphoma B-cells was selected (Mallikaratchy et al., 2007). To identify a protein target on the cell surface the aptamer was labeled by means of biotin at the 3 end of the oligonucleotide chain. Labeled TD05 in complex with a protein target was isolated from cell lysate by means of streptavidin immobilized on magnetic beads. The target was identified as the mu heavy chain of immunoglobulin M, the B-cell antigen receptor. TD05 also reacts with serum IgM molecules, an obstacle for applying the aptamer for diagnostic and therapeutic purposes in vivo. However, multimeric TD05 molecules react only with membrane-bound IgM (mIgM) which has an additional sequence, a tailpiece, nearly all which is inserted into the cell membrane with the exception of its 15 amino-acid section, which remains outside. In soluble IgM, this tailpiece is absent. Trimeric and tetrameric TD05 were prepared using a polyethyleneglycol linker (Mallikaratchy et al., 2011). Multimeric TD05 effectively combines with mIgM, a marker of Burkitt’s lymphoma cells, with increased avidity, thereby enabling the use of TD05 for targeting lymphoma cells in the body. Most likely, the external portion of the mu chain tailpiece forms part of the epitope recognized by multimeric TD05. 3.3. Immunoglobulin E During the past two decades several studies were performed on the interactions of immunoglobulin E and corresponding DNA and RNA aptamers that reacted with IgE with high affinity. The nature of the aptamer reaction sites on IgE molecule remains unclear but functional studies imply that they are located on Fc. This part of IgE is responsible for the reaction of IgE molecules with the high affinity FcRI cell receptor located on mast cells and basophiles (Drinkwater et al., 2014). Wiegand et al. (1996) demonstrated that DNA and RNA aptamers can block this reaction, a most important step in eliciting allergic responses as, following that reaction, activated cells release mediators that elicit complex allergic symptoms. Aptamers blocking IgE–FcRI interactions could serve as efficient inhibitors of such reactions. The IgE concentrations in the blood of healthy individuals are low, but in allergic patients IgE levels sharply rise. Therefore, a high IgE concentration is an important clinical marker that points towered the presence of atopic pathology. Several laboratories
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have developed aptamer assays based on various physico-chemical methods, for the measurements of the IgE concentrations. A DNA aptamer coupled with fluorescein was applied to detect IgE by means of capillary electrophoresis with laser-induced fluorescence (German et al., 1998). The method is very sensitive (detection limit 46 pM), and efficient; the results are readily reproducible. The presence of other serum proteins did not influence the measurements. Technical details of this method were expanded upon later (Buchanan et al., 2003). For evaluation of IgE concentrations by fluorescence polarization, a DNA aptamer conjugated with fluorophores fluorescein or Texas Red was used (Gokulrangan et al., 2005). This assay is very sensitive, specific, rapid and simple, and can be performed in low-nM concentrations. A method for IgE detection with an anti-IgE DNA aptamer immobilized on goldcoated quartz crystals was also developed (Liss et al., 2002). Using the method, IgE was detected in protein mixtures with high sensitivity (0.5 nmol/L). It was determined that the aptamer molecules were placed on a solid phase in a dense well-oriented position. The immobilized aptamer was stable over several weeks. More recently an IgE-specific aptamer conjugated with gold nanoparticles was applied to detect IgE at low concentrations (Liu et al., 2015). IgE in complex with a gold-labeled aptamer was determined by chemiluminescence, using a luminol-H2 O2 system. Several other sensitive assays, involving tools such as atomic force microscopy (Jiang et al., 2003), carbon nanotube field-effect transistors (Maehashi et al., 2007), surface plasmon resonance (Wang et al., 2008), and the ˇ acoustic method (Snejdárková et al., 2008), have also been applied to the study of aptamer–IgE interactions.
4. Aptamers instead of antibodies in immunoassays Immunoassays utilize specific antibodies as their primary detection component. After aptamers were invented, attempts were made to replace antibodies with specific aptamers, bearing in mind the positive features of aptamers. One of most frequently applied immune-methods, in immunological studies as well in diagnostic medicine, is the enzyme-linked immunosorbent assay (ELISA), in which two antibodies to a target molecule are used: one is a capture antibody immobilized on a microtiter plate and the second is a detection antibody, labeled by an enzyme or a fluorophore. The target molecule is located between these two antibody molecules. Several variations on this assay, in which one or both antibodies in the sandwich variants were replaced with aptamers of the same specificity have been described (Toh et al., 2015). In direct variants, target molecules are immobilized on an insoluble phase. To detect vascular endothelial growth factor (VEGV), for example, an RNA aptamer labeled by fluorescein was used as a detection reagent (Drolet et al., 1996). The capture molecule consisted of an antibody against the factor. This assay was used to determine the presence of VEGV in culture medium and in plasma, and the results was comparable with those obtained by standard ELISA. A DNA aptamer-antibody sandwich ELISA was used successfully to test for levels of MUC1 mucin, a tumor marker (Ferreira et al., 2008). This glycoprotein is expressed by adenocarcinoma cells, and can be found in the blood of cancer patients. The biotinylated aptamer specific to MUC1 and used as a capture reagent was immobilized on polystyrene titer plates coated with avidin or streptavidin. Following formation of the aptamer–MUC1 complex, a mouse anti-MUC1 antibody was added. Goat anti-mouse IgG coupled with alkaline phosphatase was used to detect the “sandwich”. After addition of p-nitrophenyl phosphate as a substrate, the intensity of the color that appeared was measured. This assay is sensitive (detection limit 1 g/ml) and could be used as a diagnostic method to detect the presence of the MUC1 tumor marker.
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For the study interactions of Leishmania histones with corresponding DNA aptamers, bacterial histones were directly immobilized on microplates (Martin et al., 2013). In one series of experiments, a digoxigenin-labeled aptamer was used as a detection reagent. An anti-digoxigenin antibody labeled with horse-radish peroxidase was added to the complex for aptamer detection. In other experiments, a biotin-labeled DNA aptamer was applied. DNA aptamers could also be used for affinity purification of bacterial proteins from cell lysates. An ELISA assay was developed to detect IgE (Wang et al., 2008). A DNA aptamer specific for IgE was immobilized on gold-covered plates as a capture reagent. As a detection reagent the fluorophore-labeled aptamer was used. Antibodies have also replaced by aptamers in lateral flow immunoassays (immunochromatographic assays or strip tests) (Chen and Yang, 2015). The tests are relatively simple and can be performed outside the laboratory. Several variants of the test have been proposed. The strips used in the assay are built from several pads, located one after the other, on a plastic plate. The detection (test) pad consists of a nitrocellulose membrane piece on which molecules for capturing targets are located. As antibodies are relatively unstable, aptamers are far more preferable as recognition molecules, in this instance. Aptamer used in this test are usually labeled by gold particles or fluorophores. Gold-labeled aptamers enable registering the results of the reaction with the naked eye, without the need for additional devices (Liu et al., 2006; Xu et al., 2009; Wang et al., 2011). In particular this assay has been applied to detect cancer cell (Liu et al., 2009).
5. Discussion During the twenty-five years that have passed since the invention of the SELEX technique, thousands of studies have been performed using various aptamers. Intensive investigations into aptamers were undertaken in university laboratories and as in specialized companies as well. Studies have been devoted to methodological innovations, usage of aptamers in various areas of biology and medicine, the preparation of new aptamer variants with more effective properties, and the use of aptamers for analytical purposes (Tombelli et al., 2005, 2007; Mairal et al., 2008; Iliuk et al., 2011; Li et al., 2014; Diafa and Hollenstein, 2015). Longer multivalent aptamers, composed of up to 200 bases, are among the new types of such reagents. They contain more binding regions, and can react more effectively with their targets, including small molecules (Bruno, 2013; Carrasquilla et al., 2015). To construct new types of aptamers, unnatural or modified bases, or aptamers with modified phosphate and sugar moieties, have been employed. The novel types of aptamers known as SOMAmers, with chemically modified nucleotides, have slow dissociation rates (Lollo et al., 2014). According to an X-ray crystal structural study of a SOMAmer in complex with a target protein, nearly all modified bases were involved in complex formation. SOMAmers have been put to use in SOMAscans, a new developed technology that enables the measurement of more than a thousand proteins in small volumes of biological samples (Ostroff et al., 2010; Webber et al., 2014). This non-invasive method has been applied to the diagnosis of lung cancer (Mehan et al., 2013, 2014). Another new methodology, termed SELMA enables the creation of DNA aptamers with modified carbohydrates (Temme and Krauss, 2015). Immobilized antigen and antibodies on surface of various solid phases have been in use by immunologists for decades (Silman and Katchalski, 1966). In the past, erythrocyte stroma and some resins (Kabat and Mayer, 1961), modified cellulose (Gurvich, 1964), Sepharose (Porath, 1974), and other materials were employed as insoluble supports. Immunosorbents were successfully used to isolate, as well as quantify, antibodies and antigens (Nezlin, 1979,
1998). To accomplish the same goals, numerous studies involving immobilization of aptamers on insoluble supports (apta-sorbents) were performed over the past decades (for review, see Acquah et al., 2015; Pichon et al., 2015). In that connection, various materials were used as solid phases; for immobilization, covalent or simple adsorption procedures were applied. Since aptamers are small molecules, their density on the support surfaces is much higher than that of immobilized protein molecules. In addition, aptamers with two or even more specificities could be located on a single support, thus providing further analytical opportunities. Apta-sorbents have been utilized effectively in immunoassays. Specific anti-IgE aptamers immobilized on gold-coated quartz crystals or gold particles were applied to measurements of IgE concentration, a parameter important in allergy diagnostics. Aptamers have successfully replaced antibodies in several classical immune methods such as ELISA and strip tests, in both cases, using immobilized aptamers. The future direction of aptamer development lies in its ability to detect cell surface proteins. In the second section of this review the use of dual apta-sorbents to identify cancer cells was described. Aptamers specific for the CD4 cell protein and labeled with fluorescein were as effective in phenotyping cells from bone marrow and lymph nodes as anti-CD4 antibodies (Zhang et al., 2010). Aptamers to other surface proteins were prepared and used for the efficient histochemical analysis of cells (Tang et al., 2007). The anti-cancer activity of aptamers specific to cell receptors has been also described (Ma et al., 2013; Mahlknecht et al., 2015; Reinemann and Strehlitz, 2014). The development of assays involving aptamers is progressing rapidly. For preparation of apta-sorbents, new solid phases have been suggested. Novel methodologies using aptamers for immunoglobulin detection, and the application of aptamers rather than antibodies in classical immunoassays have also been successfully developed. Clearly, progress in the use of aptamers will continue, taking advantage of their unique properties.
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Review
Use of aptamers in immunoassays Roald Nezlin ∗ Department of Immunology, Weizmann Institute of Science, Rehovot 7610001, Israel
a r t i c l e
i n f o
Article history: Received 21 October 2015 Received in revised form 2 December 2015 Accepted 15 December 2015 Keywords: Aptamers Apta-sorbents Ig detection Aptamers instead of antibodies
a b s t r a c t Aptamers, short single-chain DNA or RNA oligonucleotides, react specifically with small molecules, as well as with proteins. Unlike antibodies, they may be obtained relatively easily. Aptamers are now widely employed in immunological studies and could replace antibodies in immunoassays. In this short review, methods for immobilizing aptamers on various insoluble materials (so-called apta-sorbents) are described. Recent findings on their use in the detection and isolation of immunoglobulins and their application in various immunoassays are also discussed. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction For a long time immunological methods have been applied in various areas of biological and clinical research. Serum proteins, antibodies, form integral parts in all variants of immunoassays. These large protein molecules are highly specific and react with strong affinity with broad array of ligands. However, the production of antibodies is a costly, time-consuming process. This especially holds true for monoclonal antibodies. Furthermore, antibodies easily lose their specific activity after denaturation; therefore require mild conditions during experimentation. Aptamers are short, synthetic single chain DNA or RNA oligonucleotides which fold into unique three-dimensional structures. They react specifically, and with high affinity, with various ligands, low-molecular weight molecules as well as with proteins. Methods to prepare aptamers in 1990 by means of SELEX procedure were described (Tuerk and Gold, 1990; Ellington and Szostak, 1990). During the subsequent 25 years, aptamers have been widely used in various areas of biological research, including immunological studies (Nezlin, 2014), and intensively applied in the development of many effective analytical methods (Mairal et al., 2008; Iliuk et al., 2011; Li et al., 2014). The importance of aptamers in a wide range of applications of is based on several factors, among them the following: the production of aptamers is relatively cheap; aptamers are stable products and after denaturation aptamer molecules can be rapidly regenerated, with no loss of specificity; aptamers can
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be modified, particularly by conjugation with various functional groups such as fluorophores or biotin, without loss of their specific activity. This review summarizes the ways in which aptamers can be applied in various methodologies commonly used in immunological research. Immobilization of aptamers on insoluble carriers is examined, followed by descriptions of the aptamer application aimed at the detection and isolation of immunoglobulins. Furthermore, some widely applied techniques that entail the application of aptamers are discussed. 2. Immobilization of aptamers on insoluble carriers (apta-sorbents) Following the advent of aptamers, many means to immobilize them were suggested. Various insoluble supports were utilized, among them gold nanoparticles (Reinemann and Strehlitz, 2014; Sassolas et al., 2011), porous silicon (Urmann et al., 2015), glass (Huy et al., 2011) and magnetic beads (Centi et al., 2008; Kökpinar et al., 2011; Deng et al., 2014; Zhu and Walter, 2015; Ozalp et al., 2015), carbon and silica nanoparticles (Lin et al., 2014; Jo et al., 2015), and graphene (Ping et al., 2015) among others (Table 1). Two types of aptamer immobilization—non-covalent or covalent—have been used. The first variant is the simpler of the two, is based on the ability of aptamers to adsorb on the surface of a solid phase. The negative charges of sugar–phosphate backbone facilitate aptamer adsorption (Li et al., 2014). Modifications of aptamers as well as solid surfaces could be correspondingly performed. Covalent coupling is preferable as stable immobilization is important in many assays, among them chromatographic
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Table 1 Some insoluble carriers used for aptamer immobilization. Gold nanoparticles Silica nanoparticles Carbon nanoparticles Magnetic beads Glass beads Glass slides Graphene Porous silicon Tresyl resin Activated Sepharose Oxidized cellulose particles Oxidized paper
techniques. Physically adsorbed aptamer molecules are linked to a solid surface in random fashion; hence, not all of them are accessible to ligand. Covalently immobilized aptamers are arranged on solid matrix at optimal orientations. For covalent immobilization terminal nucleotides are modified and coupled with an appropriated solid surface. Such an approach was utilized for example, to immobilize of cyanuric-activated aptamers on amino-modified glass slides (Walter et al., 2008). The three-dimensional structure of aptamers is more likely to fold correctly to enable their coupling with a target, if a linker is introduced as a spacer between aptamer molecules and the surface. Sequence of thymidine nucleotides or polyethylenglycol is often used as linkers for immobilization of aptamers on gold or magnetic beads (Balamurugan et al., 2008a; Zhu and Walter, 2015). During the past decade immobilization of aptamers on gold plates or on gold nanoparticles was applied in many studies. For coupling with gold, thiol-alkane group is linked to an oligonucleotide chain terminal. This simple procedure involves arrangement of an aptamer monolayer in an orderly fashion. The resulting apta-sorbents are stable. Following addition of target molecules aggregated gold nanoparticles with immobilized aptamers change color from red to pinkish/purple and the aptamer–target interaction can be registered by the naked eye with no need for any special equipment (Elghanian et al., 1997; Storhoff et al., 1998). Surfaces such as glass slides or particles, magnetic beads and gold with acidic groups are useful platforms for the immobilization of modified aptamers. For covalent linkage several chemical groups on surfaces are used, among them are hydroxyl, amine, and carboxylic acid groups which could be further modified. An example of such procedure is the immobilization of aptamers activated with cyanuric chloride on amino-modified glass slides (Walter et al., 2008). Fine cellulose particles (Gurvich and Lechtzind, 1982) or paper (Nezlin, 2005) oxidized by NaIO4 to introduce aldehyde groups could be applied to immobilize aptamers covalently through their amino-terminal groups. A suspension of fine cellulose particles can be stored in water at 4◦ for prolonged time. Pieces of oxidized paper can be employed in strip tests. As aptamers are small molecules, their density on solid surfaces is significantly higher than that of immobilized protein molecules. On a given solid matrix it is possible to fix aptamers with different specificities simultaneously; for example, directed at two different epitopes of a single protein molecule or at two separate molecules. In some assays, such types of interactions can achieve higher levels of sensitivity (Deng et al., 2014). Dual aptamer-silica nanoparticles have been applied as effective diagnostic probes in instances of breast cancer (Jo et al., 2015). One aptamer recognized the mucin 1 cell line and a second, the epidermal growth factor cell line. Such a dual aptamer system was more effective than single aptamer particles for diagnosis of breast cancer. Moreover, the sensitivity of
the diagnostic probe was very high with a detection limit of one cell/100 l. Apta-sorbents have been successfully employed in various methodologies, including immunoassays, as well as in many techniques for the detection and extraction of various analytes in affinity chromatography, affinity capillary electrophoresis, and electrochromatography, among others (Romig et al., 1999; Giovannoli et al., 2008; Balamurugan et al., 2008b; Pichon et al., 2015). 3. Detection and isolation of immunoglobulins Over the past decade several aptamers specific to immunoglobulins (Ig) of human, mouse and rabbit origin were obtained. Interest in this problem is quite understandable, as Ig-specific aptamers after immobilization could be applied not only to detect Igs but also to the industrial-scale isolation of therapeutic antibodies (Ramsland et al., 2015). 3.1. Immunoglobulin G A 23-long RNA aptamer, Apt8-2, with the ability to interact specifically and with high affinity with the Fc region of human IgG was prepared. IgGs of animal species do not react with this aptamer. For the aptamer’s reaction with Fc␥, the GGUGCU bulging motif was found to be indispensable (Miyakawa et al., 2008). The specific aptamer reactive structure is supported by the presence of the divalent ions (Ca2+ and Mg2+ ), without which no reaction with IgG was observed. The structure of the RNA aptamer in complex with the human IgG1 molecule was studied by X-ray crystallography at 2.15 A´˚ resolution (Sugiyama et al., 2008; Miyakawa et al., 2008; Nomura et al., 2010). The RNA aptamer combines with the Fc part of IgG1 at the same site as bacterial protein A; both reagents compete with each other for the reaction with Fc␥. The Fc␥ cell receptor binding site does not overlap with the aptamer and protein A sites. Several non-electrostatic forces, such as hydrogen bonds and van der Waals forces, were shown to be involved in the aptamer–Fc interaction. Three sites on the surface of human Fc␥ are important for its interaction with the aptamer. They include amino-acid residues 316–324; 338–344; and 397–406. Among them residues 315, 342 and 404 are specific for human Fc␥. The spacial structure of human Fc␥ recognition sites is also differs from corresponding regions of animal IgGs. For preparation of apta-sorbents, the RNA aptamers were modified by attaching amino groups, via a linker to 3 or 5 ends (Miyakawa et al., 2008). The modified aptamers were immobilized on Tresyl resin or NHS-activated Sepharose. These adsorbents effectively bound IgG from human serum, as well as therapeutic chimeric or humanized antibodies from cell culture medium. Bound proteins were eluted by neutral buffer containing 10 mM EDTA. This chelating agent changes the aptamer structure by removing divalent ions and disrupts aptamer–IgG complex. The apta-sorbent could be regenerated many times by means of 6 M urea without loss of adsorbent capacity. Furthermore, preparation of the apta-sorbent is cheaper than that of the protein A-sorbent, an advantage most important for large-scale production of therapeutical antibodies. Aptamers specific to rabbit and mouse IgG were also obtained. The RNA aptamer R 18 specifically bound to native rabbit IgG but not to SDS-denatured rabbit IgG or to IgG of mouse or goat (Yoshida et al., 2008). R 18 also combined with isolated rabbit Fc␥; however the binding was significantly less effective than that with an intact IgG molecule. Two stretches of R 18, each three nucleotides long, are
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critical to its binding to the IgG molecule. It has been proposed that nearly the entire R 18 molecule combines with IgG. Mg2+ ions are required for the effective reaction of R 18 with IgG. Removal of these ions with EDTA resulted in dissociation of the R 18–IgG complex after which the aptamer could be again effectively used for purification of rabbit antibodies. The biotinylated R 18 was successfully used in blotting experiments as a secondary anti-rabbit IgG reagent. DNA and RNA aptamers that specifically reacted with mouse Fc␥ were prepared. The RNA aptamer MIG1 was found to specifically interact with the Fc region of three mouse IgG subclasses—IgG1, IgG2a and IgG3 (Sakai et al., 2008). Denatured IgG molecules, on the other hand, were unable to combine with the aptamer. IgG of four other species did not react with MIG-1, with the exception of rat IgG which cross-reacted with mouse IgG due to similarities in their amino-acid sequences. MIG-1 effectively recognized mouse IgG in blotting analysis. DNA aptamers that reacted with mouse IgG1 and IgG2a were isolated, using a modified SELEX procedure (Ma et al., 2013). These aptamers display a similar stem-loop secondary structure which is important for binding. Dot blot and electrophoretic mobility shift (EMSA) assays were utilized to study the binding activity of the aptamers. 3.2. Immunoglobulin M A DNA aptamer TD05 which specifically recognizes an epitope expressed on B lymphocytes and Burkitt’s lymphoma B-cells was selected (Mallikaratchy et al., 2007). To identify a protein target on the cell surface the aptamer was labeled by means of biotin at the 3 end of the oligonucleotide chain. Labeled TD05 in complex with a protein target was isolated from cell lysate by means of streptavidin immobilized on magnetic beads. The target was identified as the mu heavy chain of immunoglobulin M, the B-cell antigen receptor. TD05 also reacts with serum IgM molecules, an obstacle for applying the aptamer for diagnostic and therapeutic purposes in vivo. However, multimeric TD05 molecules react only with membrane-bound IgM (mIgM) which has an additional sequence, a tailpiece, nearly all which is inserted into the cell membrane with the exception of its 15 amino-acid section, which remains outside. In soluble IgM, this tailpiece is absent. Trimeric and tetrameric TD05 were prepared using a polyethyleneglycol linker (Mallikaratchy et al., 2011). Multimeric TD05 effectively combines with mIgM, a marker of Burkitt’s lymphoma cells, with increased avidity, thereby enabling the use of TD05 for targeting lymphoma cells in the body. Most likely, the external portion of the mu chain tailpiece forms part of the epitope recognized by multimeric TD05. 3.3. Immunoglobulin E During the past two decades several studies were performed on the interactions of immunoglobulin E and corresponding DNA and RNA aptamers that reacted with IgE with high affinity. The nature of the aptamer reaction sites on IgE molecule remains unclear but functional studies imply that they are located on Fc. This part of IgE is responsible for the reaction of IgE molecules with the high affinity FcRI cell receptor located on mast cells and basophiles (Drinkwater et al., 2014). Wiegand et al. (1996) demonstrated that DNA and RNA aptamers can block this reaction, a most important step in eliciting allergic responses as, following that reaction, activated cells release mediators that elicit complex allergic symptoms. Aptamers blocking IgE–FcRI interactions could serve as efficient inhibitors of such reactions. The IgE concentrations in the blood of healthy individuals are low, but in allergic patients IgE levels sharply rise. Therefore, a high IgE concentration is an important clinical marker that points towered the presence of atopic pathology. Several laboratories
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have developed aptamer assays based on various physico-chemical methods, for the measurements of the IgE concentrations. A DNA aptamer coupled with fluorescein was applied to detect IgE by means of capillary electrophoresis with laser-induced fluorescence (German et al., 1998). The method is very sensitive (detection limit 46 pM), and efficient; the results are readily reproducible. The presence of other serum proteins did not influence the measurements. Technical details of this method were expanded upon later (Buchanan et al., 2003). For evaluation of IgE concentrations by fluorescence polarization, a DNA aptamer conjugated with fluorophores fluorescein or Texas Red was used (Gokulrangan et al., 2005). This assay is very sensitive, specific, rapid and simple, and can be performed in low-nM concentrations. A method for IgE detection with an anti-IgE DNA aptamer immobilized on goldcoated quartz crystals was also developed (Liss et al., 2002). Using the method, IgE was detected in protein mixtures with high sensitivity (0.5 nmol/L). It was determined that the aptamer molecules were placed on a solid phase in a dense well-oriented position. The immobilized aptamer was stable over several weeks. More recently an IgE-specific aptamer conjugated with gold nanoparticles was applied to detect IgE at low concentrations (Liu et al., 2015). IgE in complex with a gold-labeled aptamer was determined by chemiluminescence, using a luminol-H2 O2 system. Several other sensitive assays, involving tools such as atomic force microscopy (Jiang et al., 2003), carbon nanotube field-effect transistors (Maehashi et al., 2007), surface plasmon resonance (Wang et al., 2008), and the ˇ acoustic method (Snejdárková et al., 2008), have also been applied to the study of aptamer–IgE interactions.
4. Aptamers instead of antibodies in immunoassays Immunoassays utilize specific antibodies as their primary detection component. After aptamers were invented, attempts were made to replace antibodies with specific aptamers, bearing in mind the positive features of aptamers. One of most frequently applied immune-methods, in immunological studies as well in diagnostic medicine, is the enzyme-linked immunosorbent assay (ELISA), in which two antibodies to a target molecule are used: one is a capture antibody immobilized on a microtiter plate and the second is a detection antibody, labeled by an enzyme or a fluorophore. The target molecule is located between these two antibody molecules. Several variations on this assay, in which one or both antibodies in the sandwich variants were replaced with aptamers of the same specificity have been described (Toh et al., 2015). In direct variants, target molecules are immobilized on an insoluble phase. To detect vascular endothelial growth factor (VEGV), for example, an RNA aptamer labeled by fluorescein was used as a detection reagent (Drolet et al., 1996). The capture molecule consisted of an antibody against the factor. This assay was used to determine the presence of VEGV in culture medium and in plasma, and the results was comparable with those obtained by standard ELISA. A DNA aptamer-antibody sandwich ELISA was used successfully to test for levels of MUC1 mucin, a tumor marker (Ferreira et al., 2008). This glycoprotein is expressed by adenocarcinoma cells, and can be found in the blood of cancer patients. The biotinylated aptamer specific to MUC1 and used as a capture reagent was immobilized on polystyrene titer plates coated with avidin or streptavidin. Following formation of the aptamer–MUC1 complex, a mouse anti-MUC1 antibody was added. Goat anti-mouse IgG coupled with alkaline phosphatase was used to detect the “sandwich”. After addition of p-nitrophenyl phosphate as a substrate, the intensity of the color that appeared was measured. This assay is sensitive (detection limit 1 g/ml) and could be used as a diagnostic method to detect the presence of the MUC1 tumor marker.
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For the study interactions of Leishmania histones with corresponding DNA aptamers, bacterial histones were directly immobilized on microplates (Martin et al., 2013). In one series of experiments, a digoxigenin-labeled aptamer was used as a detection reagent. An anti-digoxigenin antibody labeled with horse-radish peroxidase was added to the complex for aptamer detection. In other experiments, a biotin-labeled DNA aptamer was applied. DNA aptamers could also be used for affinity purification of bacterial proteins from cell lysates. An ELISA assay was developed to detect IgE (Wang et al., 2008). A DNA aptamer specific for IgE was immobilized on gold-covered plates as a capture reagent. As a detection reagent the fluorophore-labeled aptamer was used. Antibodies have also replaced by aptamers in lateral flow immunoassays (immunochromatographic assays or strip tests) (Chen and Yang, 2015). The tests are relatively simple and can be performed outside the laboratory. Several variants of the test have been proposed. The strips used in the assay are built from several pads, located one after the other, on a plastic plate. The detection (test) pad consists of a nitrocellulose membrane piece on which molecules for capturing targets are located. As antibodies are relatively unstable, aptamers are far more preferable as recognition molecules, in this instance. Aptamer used in this test are usually labeled by gold particles or fluorophores. Gold-labeled aptamers enable registering the results of the reaction with the naked eye, without the need for additional devices (Liu et al., 2006; Xu et al., 2009; Wang et al., 2011). In particular this assay has been applied to detect cancer cell (Liu et al., 2009).
5. Discussion During the twenty-five years that have passed since the invention of the SELEX technique, thousands of studies have been performed using various aptamers. Intensive investigations into aptamers were undertaken in university laboratories and as in specialized companies as well. Studies have been devoted to methodological innovations, usage of aptamers in various areas of biology and medicine, the preparation of new aptamer variants with more effective properties, and the use of aptamers for analytical purposes (Tombelli et al., 2005, 2007; Mairal et al., 2008; Iliuk et al., 2011; Li et al., 2014; Diafa and Hollenstein, 2015). Longer multivalent aptamers, composed of up to 200 bases, are among the new types of such reagents. They contain more binding regions, and can react more effectively with their targets, including small molecules (Bruno, 2013; Carrasquilla et al., 2015). To construct new types of aptamers, unnatural or modified bases, or aptamers with modified phosphate and sugar moieties, have been employed. The novel types of aptamers known as SOMAmers, with chemically modified nucleotides, have slow dissociation rates (Lollo et al., 2014). According to an X-ray crystal structural study of a SOMAmer in complex with a target protein, nearly all modified bases were involved in complex formation. SOMAmers have been put to use in SOMAscans, a new developed technology that enables the measurement of more than a thousand proteins in small volumes of biological samples (Ostroff et al., 2010; Webber et al., 2014). This non-invasive method has been applied to the diagnosis of lung cancer (Mehan et al., 2013, 2014). Another new methodology, termed SELMA enables the creation of DNA aptamers with modified carbohydrates (Temme and Krauss, 2015). Immobilized antigen and antibodies on surface of various solid phases have been in use by immunologists for decades (Silman and Katchalski, 1966). In the past, erythrocyte stroma and some resins (Kabat and Mayer, 1961), modified cellulose (Gurvich, 1964), Sepharose (Porath, 1974), and other materials were employed as insoluble supports. Immunosorbents were successfully used to isolate, as well as quantify, antibodies and antigens (Nezlin, 1979,
1998). To accomplish the same goals, numerous studies involving immobilization of aptamers on insoluble supports (apta-sorbents) were performed over the past decades (for review, see Acquah et al., 2015; Pichon et al., 2015). In that connection, various materials were used as solid phases; for immobilization, covalent or simple adsorption procedures were applied. Since aptamers are small molecules, their density on the support surfaces is much higher than that of immobilized protein molecules. In addition, aptamers with two or even more specificities could be located on a single support, thus providing further analytical opportunities. Apta-sorbents have been utilized effectively in immunoassays. Specific anti-IgE aptamers immobilized on gold-coated quartz crystals or gold particles were applied to measurements of IgE concentration, a parameter important in allergy diagnostics. Aptamers have successfully replaced antibodies in several classical immune methods such as ELISA and strip tests, in both cases, using immobilized aptamers. The future direction of aptamer development lies in its ability to detect cell surface proteins. In the second section of this review the use of dual apta-sorbents to identify cancer cells was described. Aptamers specific for the CD4 cell protein and labeled with fluorescein were as effective in phenotyping cells from bone marrow and lymph nodes as anti-CD4 antibodies (Zhang et al., 2010). Aptamers to other surface proteins were prepared and used for the efficient histochemical analysis of cells (Tang et al., 2007). The anti-cancer activity of aptamers specific to cell receptors has been also described (Ma et al., 2013; Mahlknecht et al., 2015; Reinemann and Strehlitz, 2014). The development of assays involving aptamers is progressing rapidly. For preparation of apta-sorbents, new solid phases have been suggested. Novel methodologies using aptamers for immunoglobulin detection, and the application of aptamers rather than antibodies in classical immunoassays have also been successfully developed. Clearly, progress in the use of aptamers will continue, taking advantage of their unique properties.
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