Robustness, stability, and ease of use may soon bring aptamers to the forefront of detection technology.
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A conceptualized change in the three-dimensional structure of a DNA sequence in response to a target protein. (Image courtesy Rational Systems LLC)
Research stemming from the Human Genome Project has increased the demand for and applications of DNA sensors. Several commercial platforms are now available, including those of Affymetrix (Santa Clara, CA) and Illumina (San Diego). However, the DNA sequence is of limited use for diagnostic purposes because it presents a static picture of only one of the many ways in which a cell might express proteins in its dynamic life process. Genetic information is valued for its ability to predict a phenotype, while RNA translation leads to differential protein expression. The ultimate functional form is often determined by posttranslational modification of the proteins. Thus, it is difficult to assess the functionality of the ultimate gene product from the DNA level.
It is often necessary to directly detect proteins instead of DNA in order to fully understand cellular function, detect diseases, monitor the progression of existing diseases, or detect a drug's effect in an organism.
Investigators select detection technologies based on the target of interest and whether they are working at the genetic or protein level. Microarray technology allows for a large number of DNA sequences to be analyzed simultaneously. However, antibody-based strategies predominate for selective protein detection, and antibody-based microarrays are being developed for the simultaneous analysis of a large number of proteins.
Randox Laboratories (Antrim, UK) launched the first protein detection array in 2003. However, microarrays have not been used successfully for protein detection because researchers have not been able to create a large antibody pool for array-based detection.
This article looks at applying aptamers (i.e., oligonucleotides used in DNA arrays) to protein detection. Aptamers have been shown to bind not only with their complementary oligonucleotide strands but also with virtually any target.
Anatomy of an Aptamer
Aptamers are small (i.e., 40 to 100 bases), synthetic oligonucleotides that can specifically recognize and bind to virtually any kind of target, including ions, whole cells, drugs, toxins, low-molecular-weight ligands, peptides, and proteins.
An aptamer's affinity depends on its target type. Aptamers against small molecules have affinities in the micromolar range (e.g., 2.8 mM for dopamine and ATP 6 mM for adenosine 5 triphosphate).1 Aptamers have shown affinities in the nanomolar and subnanomolar range against some proteins, such as vascular endothelial growth factor with an affinity of 100 pM, and keratinocyte growth factor, 1 pM.2
High affinity often means high specificity for aptamers. They can discriminate between targets using subtle structural differences such as the presence of a hydroxyl group or a methyl group.2 Furthermore, they can distinguish between enzymes with similar catalytic function, such as a-thrombin and g-thrombin.3 Impressively, aptamers can also distinguish between enantiomers.
A higher degree of specificity has been demonstrated with aptamers than with antibody preparations. For example, aptamers provided high molecular discrimination between theophylline and caffeine, which differ only in a methyl group.4 In fact, the binding affinity for the theophylline aptamer was tenfold higher than for the caffeine aptamer. In addition, when antibodies were used as the biorecognition element, caffeine produced significant interferences in the detection of theophylline.5
Origins of Aptamers
Aptamers emerged from the experimental efforts of three independent groups that first published their work in 1990. Gerald F. Joyce's group at the Scripps Research Institute (La Jolla, CA) was looking for new enzymatic activity of RNA.6 The researchers used in vitro mutation, selection, and amplification to isolate the RNA with enzymatic functionality. This approach became the basis for the current in vitro selection of aptamers.
Larry Gold's group at the University of Colorado (Denver) was trying to identify the sequences of T4 DNA polymerase in vitro. Their library was based on the natural structure, but included a randomized eight-nucleotide sequence. The group named the patented process (U.S. Patent 5,270,163) of in vitro selection “Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The SELEX process could identify the most selective aptamer for an enzyme.
A month after this discovery, Jack W. Szostak and Andrew D. Ellington at the Massachusetts General Hospital (Boston) reported the use of in vitro selection to isolate molecules with specific ligand-binding activities. They created a library with 100 nucleotides of randomized sequences that were unrelated to any known oligonucleotide sequence. The targets were organic dyes without known nucleic acid ligands. This group coined the term aptamer, which comes from the Latin aptus, meaning “to fit.”
Figure 1. (click to enlarge) Scheme of the schematic evolution of ligands by exponential enrichment (SELEX) process.
SELEX is a technique used for isolating functional synthetic nucleic acids. It allows for the in vitro screening of large, random libraries of oligonucleotides by an iterative process of adsorption, recovery, and amplification of the oligonucleotide sequences (see Figure 1). A database of primary and derived data from high-throughput SELEX experiments can be accessed for free online at www.isrec.isb-sib.ch/htpselex.
The SELEX process starts with the synthesis of a library of single-stranded oligonucleotides. The first round of a typical SELEX will contain from 1014 to 1015 unique sequences. The chemically synthesized single-strand (ss) DNA are then amplified by polymerase chain reaction (PCR) in order to generate the corresponding double-stranded DNA. Depending on the aptamer desired, the library is converted into either ssDNA by strand separation or RNA by in vitro transcription. The ssDNA are then incubated with the target of interest. The nucleic acids adopting a conformation that allows them to bind with the target are separated by filtration through a nitrocellulose column or an affinity column (for small molecules). The aptamer-target duplexes are eluted and amplified by reverse transcriptase- PCR, in the case of RNA aptamers, or PCR in the case of DNA aptamers. This creates a smaller enriched pool of molecules for the next cycle. The process requires from 8 to 15 iterations of the cycle, which are carried out under increasingly stringent conditions to achieve an aptamer of high affinity.
Aptamers versus Antibodies
Table I. (click to enlarge) Advantages of aptamers as compared to antibodies.
Aptamers, the nucleic acid equivalent of antibodies, possess characteristics that make them superior to antibodies when used as sensing elements for proteomic sensors (see Table I). The antibody identification process depends on an animal host. The expression process is further complicated when the targets are toxins, which can harm the host, or low-molecular-weight molecules, which trigger a minimal immunogenic response. An antibody's performance is lot specific, and although monoclonal preparations have reduced the lot-to-lot variability associated, the preparation still requires a lengthy purification protocol.
In contrast, aptamers are designed in vitro and are thus independent of any animal host. Aptamers are identified using automated oligonucleotide synthesis and screening systems. The binding specificity, affinity, and stability of aptamers can be improved by molecular evolution techniques or by rational design. Additionally, the aptamer identification process can be changed to obtain an aptamer that interacts with specific target regions or under a differing binding condition. Once identified, aptamers are readily created with extremely high reproducibility and purity, using established chemical synthesis processes.
Aptamers can also be labeled with a reporter molecule without affecting their affinity by employing the protocols already established for oligonucleotide labeling.7 An aptamer sensor can operate in a wide variety of sample matrices, including nonphysiological buffers and temperature conditions that would denature typical antibody formulations. Kinetic parameters in an aptamer interaction can be changed on demand. Antibodies are large and complex molecules that are sensitive to nonphysiological pH and temperature, which can limit their use in reusable sensors. However, aptamers can undergo repeated cycles of denaturation and renaturation without damaging their structure. Furthermore, they can be transported at ambient temperatures without degradation, stored for long duration prior to use, and subjected to numerous freeze-and-thaw cycles.
Applications of Aptamers
Aptamers were first applied in the field of therapeutic drugs because aptamers can block receptors and inhibit protein activity with high affinity and specificity. Aptamers have also been used to validate drug targets and screen drug candidates. Their advantages of small size, quick elimination, low production cost, biocompatibility, biodegradability, and no cross-reactivity with antibody binding receptors make them good candidates for therapeutic applications. However, because blood is rich in nucleases, the aptamer therapeutic must be modified to avoid nuclease attack. Spigelmers, one such modification, are composed of L-nucleotides instead of the natural D-nucleotides; thus, the biological L-nucleases cannot recognize and cleave the artificial oligonucleotides.
One of the most successful therapeutic applications of an aptamer has been in the treatment of age-related macular degeneration. Macugen (OSI Pharmaceuticals, Melville, NY) is an aptamer that inhibits antivascular endothelial growth factor, which participates in the growth of abnormal blood vessels in the eyes that cause vision loss. FDA has approved Macugen for patients with neovascular age-related macular degeneration.
ARC183 (Gilead, Foster City, CA) is another aptamer with therapeutic applications. This antithrombin aptamer inhibits most physiologic blood coagulation events and has passed Phase I clinical trials. Other examples of therapeutic applications for aptamers include an aptamer targeting C5 that inhibits human serum hemolytic activity.8 The latter inhibits the activity of a trypsin-like protease of nonstructured protein (NS3) for the control of polyprotein secretin from the hepatitis C virus.
In addition to their pharmaceutical applications, aptamers have been used in the field of separation chemistry. Affinity chromatography is used for the selection and separation of biomolecules from complex mixtures. Antibodies are frequently employed as receptor molecules in stationary phases, due to their high affinity and selectivity. Nevertheless, their application in chromatography is limited. In addition to some of the previously identified constraints (see Table I), their large molecular size limits the surface loading on the stationary phase and the affinity binding capacity of the matrix. These difficulties are further compounded by the more complex process of obtaining antibodies for small molecules.
Aptamers have shown promise for use in affinity chromatography because of their potential to overcome some of the limitations of antibodies. For example, adenosine and its analogues have been successfully purified using aptamers supported on a stationary phase.9 Furthermore, aptamers have been similarly used to separate amino acids and polycyclic aromatic hydrocarbons.10
Sensor Applications of Aptamers
Aptamers can function as the biorecognition elements in biosensor applications. Biosensors combine a biological species that selectively detects an analyte with a physical transduction method that produces a measurable signal in response to the action of the biological species. A biosensor's analytical targets are not always of a biological origin. In fact, almost any chemical analyte can be detected with biosensors.
Aptasensors are biosensors that use aptamers as their biorecognition element. Aptasensors were first used in 1996 as the selective component in an optical sensor application where they were part of a model system consisting of human-neutrophil-elastase-coated beads that interact with fluorescent-tagged aptamers.11
Eight months later, two new examples of aptasensors appeared: one in which a radiolabeled aptamer was used to detect different protein kinase C isozymes and another in which an enzyme-linked sandwich assay used a SELEX-derived fluorescently labeled oligonucleotide.12,13 They have since been applied in environmental, industrial, defense, and medical fields.
Table II. (click to enlarge) Ultrasensitive aptasensor transducers.
In the past decade, hundreds of aptasensor papers have been published, using an assortment of transductors (see Table II). These applications often use fluorescence labels. For example, L-adenosine detection was transduced by evanescent wave-induced fluorescence.14 Thrombin detection was performed with aptamer-fluorescence-quenching pairs and by a competitive assay of fluorescently labeled thrombin with thrombin on an aptamer-modified optical fiber.15
Aptamers have been used in many detection techniques. Quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) are two widely described techniques for transduction and detection of non-labeled aptamers. A label-free protein detection strategy uses a microfabricated cantilever-based sensor that is functionalized with aptamers to act as receptors of Taq DNA poly-merase.16 Atomic force microscopy (AFM) has been used to measure the specific interaction between a protein immunoglobulin E (IgE) and an aptamer.17 In addition, a surface acoustic wave biosensor array couples aptamers to detect thrombin and HIV-1 Rev peptide.18
Figure 2. (click to enlarge) An electrochemical aptamer beacon sensor for thrombin
Recently, electrochemical aptasensors have been developed.19 For example, a two-aptamer sandwich configuration has been used in a bio-sensor, and impedance spectroscopy has been used to detect a sensitive interaction of an aptamer with IgE.19,20 Aptamers have also been used as electrochemical beacons, which are oligonucleotide molecules able to undergo a measurable conformation change in response to the target analyte (see Figure 2). A ferrocene-labeled aptamer beacon sensor has been used for thrombin detection, and an electrochemical aptamer-based beacon has been developed for cocaine detection.21,22
Many different electrochemical aptasensors have been used for thrombin detection.23 In one of these configurations, thrombin was detected using a sandwich format. Sandwich assay formats are only applicable in the case of targets with multiple aptamer binding regions. Thrombin has two electropositive exosites, both of which are capable of binding a specific aptamer.
Figure 3. (click to enlarge) An electrochemical aptamer base
sensor in a sandwich format. HRP = horseradish
The sandwich assay was performed by immobilizing a thiolated aptamer as a self-assembled monolayer on a gold electrode. The electrode was subsequently incubated with thrombin. In a second incubation step, a horseradish peroxidase (HRP)–labeled aptamer was allowed to bind to the other thrombin exosite. The HRP-labeled aptamer was prepared by incubating a biotin-labeled aptamer with streptavidin-HRP, effectively forming a peroxidase-labeled aptamer. The HRP, thus immobilized at the electrode surface, was measured electrochemically using hydrogen peroxide and a diffusional, osmium-based mediator ([Os(bpy)2(pyr-CH2-NH2)]Cl). The current measured on the electrode was related to the quantity of thrombin by calibration (see Figure 3).
Figure 4. (click to enlarge) Amperometric detection of thrombin through a sandwich format, applied in an aptasensor. HRP = horseradish peroxidase.
The optimized configuration of aptamer-thrombin-aptamer interaction generated a current of 4.5 µA. Negative controls verified that the signal was not due to nonspecific adsorption. A negative control, without thrombin immobilized on the electrode surface, resulted in a current of 1.8 µA. A second negative control, without the base aptamer, produced an identical current. A third negative control, without HRP-labeled aptamer, resulted in a current of 0.2 µA (see Figure 4).
The electrochemical response was reported as the difference in current at –0.1 V (versus silver and silver chloride) before and after addition of 6 mM of hydrogen peroxide and an osmium redox mediator. Hydrogen peroxide served as the substrate for the HRP label and the osmium redox mediator allowed for the transduction of the enzyme turnover into a measurable current response.
Figure 5. (click to enlarge) Chronoamperometric response of thrombin concentrations in a sandwich aptasensor format.
While this assay suffered from significant nonspecific adsorption of HRP-labeled aptamer, the limit of detection for this system was still 92 nM (see Figure 5). The limit of detection was measured by fitting the current-versus-thrombin values that had a linear response along a linear regression line. The standard deviation of the line was measured and multiplied by 3 to reduce the probability of a false nondetection to 5%. This value was added to the value of signal that was distinguishable from noise response.
As a result of the peroxidase label, the electrochemical assay displayed a significant background response (see Figures 4 and 5). The peroxidase label exhibited strong nonspecific adsorption to the electrochemical sensor. This effect has been observed in other electrochemical affinity sensors that use peroxidase. The electrochemical detection of peroxidase is extremely sensitive, and the conditions necessary to prevent the peroxidase from binding to the electrode surface have not been sufficiently investigated.
The ability of oligonucleotide sequences to bind strongly and specifically to targets other than the obvious complementary sequence allows oligonucleotide-based detection strategies to be used in new fields, such as therapeutics, separation techniques, and biosensors. The application of aptamers to detection strategies is especially promising.
Aptamers offer effective identification and production methods (e.g., SELEX), easy chemical synthesis (e.g., automated synthesis directly on an analytical support), immobilization at high density, the possibility to detect all toxins, and stability at nonphysiological conditions. In fact, aptamers may potentially replace antibodies in biosensor applications. However, the library of aptamer biosensors will first need to match that of antibodies. Second, many readily available methods for developing, optimizing, and troubleshooting these assays will need to be created before aptamers will be readily adopted.
(left to right) Monica Mir is a recent PhD candidate in the chemical engineering department at the University Rovira i Virgili, Spain. Ioanis Katakis, PhD, is an associate professor of chemical engineering at the University Rovira i Virgili, Spain. Mark S. Vreeke, PhD, is a visiting professor at the University Rovira i Virgili, Spain and a partner at Rational Systems LLC (Houston), a business process and technical consulting firm. The authors can be reached at email@example.com, firstname.lastname@example.org, and email@example.com.
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