We have developed an improved and straightforward protocol for DNA aptamer production and have characterized aptamers that recognize TTF1, a member of the NK homeodomain transcription factors (15
). The use of aptamers as protein affinity reagents offers advantages over the use of antibodies. Nucleic acids are easily synthesized or amplified by PCR, therefore a vast supply of consistent quality is available. Also, nucleic acids can easily be modified to incorporate tags, such as biotin or fluorescent molecules, for detection and/or immobilization. Additionally, aptamers are smaller (<25 kDa) and more stable than antibodies. Moreover, unlike the requirement for milligram quantities of protein or peptide for antibody production, only microgram quantities of protein or peptide are required for aptamer SELEX. These properties, coupled to the present technology available for DNA microarrays, make aptamers very suitable for use in protein microarrays as a ligand, or for detecting proteins bound to a chip surface (21
Despite these advantages, aptamers have rarely been selected for general use since the technology was developed 13 years ago. Approximately 40 unique aptamers against proteins or peptides have been described in more than 300 references in the literature. This lack of widespread use may be attributed to challenges in adapting existing protocols to particular targets and a general lack of fine details in existing methods. Many variations in aptamer production protocols have been described in which the method of protein target partitioning seems to vary the most. Unbound molecules have been removed from target proteins via: (i) filtration on a membrane (13
); (ii) column chromatography, in which the targets are bound to a matrix, such as Sepharose, using a covalent linkage or an affinity tag (22
); and (iii) binding of the protein to the wells of a microtiter plate (23
The novelty of the protocol that we have described is the use of Ni-NTA magnetic beads for the immobilization of His6
-tagged protein targets during selection. His6
tags are widely used in recombinant protein production, for example our group has described an efficient protein production pipeline for high-throughput generation of His6
-tagged proteins in E.coli
). The use of a tag for immobilization promotes the proper orientation of proteins uniformly on a bead surface, and simultaneously provides a purification step, thereby reducing the chances of selection toward contaminants. Paramagnetic beads are an optimal solid support for parallel processing of both proteins and nucleic acids. Very small amounts of magnetic beads with proteins bound can be rapidly partitioned from unselected material, stringently washed and subsequently eluted. Others have claimed that filtration is necessary to partition the bound aptamers from unselected molecules in order to obtain sufficient stringency (14
). However, in our protocol, the washes were sufficiently stringent using magnetic beads. This is an advantage because partitioning by filtration is a cumbersome process for multiple targets but magnetic bead separations are easily accomplished in parallel, manually or automated. Additionally, we have taken advantage of the highly specific and strong streptavidin–biotin interaction for several applications: (i) generating single-stranded material from biotinylated PCR products after amplification at each round of selection using streptavidin magnetic beads; (ii) detecting biotinylated aptamers in enzyme-linked assays and western blot analysis; (iii) immobilizing biotinylated aptamers to streptavidin beads for purification of protein target; and (iv) immobilizing biotinylated aptamers to streptavidin sensor chips in BIAcore measurements.
Using manual processing (by one person), we were able to complete three rounds of selection per day on eight samples. We found that 15 rounds of selection produced high affinity aptamers. Therefore, our present throughput could be approximately 32 aptamers per month. However, our protocol is amenable to a 96-well approach and could be scaled-up to produce about 384 aptamers per month using manual processing. Others have described an automated aptamer acquisition platform with a throughput of 120/month for eight proteins; however, it requires customized robotics not available to many laboratories (14
). Another high-throughput SELEX protocol using 96-well microtiter plates has been described that is compatible with robotics and was tested manually (23
). However, that protocol relied on hydrophobic immobilization of proteins on microtiter plates, and the authors concede that the four proteins tested adhered to the wells with varying efficiency, making it difficult to control the amount of protein in each experiment. In addition, use of a hydrophobic immobilization would also result in proteins with variable orientations on the surface, reducing the effective concentration of available active sites. Lastly, since this method is non-specific, it would also result in the immobilization of all proteins in the sample, including protein contaminants that could interfere with the aptamer selection process.
We have illustrated the functional versatility of aptamers in several assays. Enzyme-linked assays provide a means of quickly evaluating a group of aptamers from a selection by measuring their relative affinities, and this kind of triage can be used to prioritize aptamers for more detailed characterization (24
). We demonstrate that enzyme-linked assays can also provide information about cross-reactivity. We are able to measure significant signals over very low background by using biotinylated aptamers to detect proteins in a 96-well microtiter plate and a peroxidase-conjugated streptavidin and colorimetric substrate to detect the bound aptamers. Enzyme-linked assays offer advantages over other techniques, such as equilibrium dialysis and electrophoretic mobility shift assays, that are used to evaluate aptamers from a selection. These advantages include the lack of radioisotope usage, increased throughput in a 96-well plate, minimization of waste and ease of precise quantitation of the relative binding affinities.
Using aptamers in a protein blot analysis is another means of characterizing their specificity. We have tested TTF1 aptamers ‘A’ and ‘C’ in chemiluminescent protein blot analysis; however, only the TTF1 aptamer ‘A’ worked in this application, suggesting that, just as some antibodies fail to recognize the denatured form of a protein, some aptamers will recognize epitopes that are absent in the denatured form of the protein. The TTF1 aptamer showed no cross-reactivity to E.coli proteins in a cleared lysate on the blot and was similar in specificity to that observed for the anti-PentaHis–HRP antibody.
The key function of high affinity aptamers in applications such as protein purification, protein profiling chips and diagnostics is to recognize and separate the target protein from a complex mixture of proteins. We have described in this work the first successful application of aptamer affinity chromatography for one-step purification of a protein from the complex mixture of proteins in the soluble fraction of bacterial cell lysates. Although aptamer affinity chromatography has been described and demonstrated for the purification of a protein from conditioned cell culture media, this purification technique has not been previously demonstrated for more complex samples such as cell lysates (25
). Detrimental effects from DNase activity in our purification from bacterial lysates were not observed, unlike the problems associated with DNase degradation of aptamers that occurs when purifying targets from serum (25
). Importantly, aptamer affinity chromatography provides a means of protein purification of the native form of a protein without relying on affinity tags that may adversely affect protein structure, function or ability to form crystals for structural characterization.
We have recently learned that TTF1 is a highly specific marker for primary lung adenocarcinomas, and antibodies against TTF1 have been recommended to be included in a panel of antibodies for the differential diagnosis between primary and metastatic adenocarcinomas of the lung (26
). We have initiated studies in our lab to determine whether or not the aptamers that we have produced against C.intestinalis
TTF1 recognize human TTF1; if so, the aptamers described here may become a valuable diagnostic tool for primary lung adenocarcinoma.