Improving aptamer selection efficiency through volume dilution, magnetic concentration, and continuous washing in microfluidic channels

The generation of nucleic acid aptamers with high affinity typically entails a time-consuming, iterative process of binding, separation, and amplification. It would therefore be beneficial to develop an efficient selection strategy that can generate these high-quality aptamers rapidly, economically, and reproducibly. Toward this goal, we have developed a method that efficiently generates DNA aptamers with slow off-rates. This methodology, called VDC-MSELEX, pairs the volume dilution challenge process with microfluidic separation for magnetic bead-assisted aptamer selection. This method offers improved aptamer selection efficiencies through the application of highly stringent selection conditions: it retrieves a small number (<10(6)) of magnetic beads suspended in a large volume (>50 mL) and concentrates them into a microfluidic chamber (8 μL) with minimal loss for continuous washing. We performed three rounds of the VDC-MSELEX using streptavidin (SA) as the target and obtained new DNA aptamer sequences with low nanomolar affinity that specifically bind to the SA proteins.     

Functional-group specific aptamers indirectly recognizing compounds with alkyl amino group

Aptamers are usually generated against a specific molecule. Their high selectivity makes them only suitable for studying specific targets. Since it is nearly impossible to generate aptamers for every molecule, it can be of great interest to select aptamers recognizing a common feature of a group of molecules in many applications. In this paper, we describe the selection of aptamers for indirect recognition of alkyl amino groups. Because amino groups are small and positive charged, we introduced a protection group, p-nitrobenzene sulfonyl (p-nosyl) to convert them into a form suitable for aptamer selection. Taking N(ε)-p-nosyl-l-lysine (PSL) as a target, we obtained a group of aptamers using the SELEX technique. Two optimized aptamers, M6b-M14 and M13a exhibit strong affinity to PSL with the K(d) values in the range of 2-5 μM. They also show strong affinity to other compounds containing p-nosyl-protected amino groups except those also possessing an α-carboxyl group. Both aptamers adopt an antiparallel G-quadruplex structure when binding to targets. An aptamer beacon based on M6b-M14 showed good selectivity toward the reaction mixture of p-nosyl-Cl and alkyl amino compounds, and could recognize lysine from amino acid mixtures indirectly, suggesting that aptamers against a common moiety of a certain type of molecules can potentially lead to many new applications. Through this study, we have demonstrated the ability to select aptamers for a specific part of an organic compound, and the chemical conversion approach may prove to be valuable for aptamer selection against molecules that are generally difficult for SELEX.     

Cancer cell targeting using multiple aptamers conjugated on nanorods

Molecular recognition toward specific cells is a key issue for effective disease, such as cancer, diagnosis and therapy. Although many molecular probes such as aptamers and antibodies can recognize the unique molecular signatures of cancer cells, some of these probes only have relatively weak binding affinities. This results in poor signaling and hinders cell targeting. Here, we use Au-Ag nanorods (NRs) as a nanoplatform for multivalent binding by multiple aptamers on the rod to increase both the signal and binding strengths of these aptamers in cancer cell recognition. Up to 80 fluorophore-labeled aptamers can be attached on a 12 nm x 56 nm NR, resulting in a much stronger fluorescence signal than that of an individual dye-labeled aptamer probe. The molecular assembly of aptamers on the NR surfaces also significantly improves the binding affinity with cancer cells through simultaneous multivalent interactions with the cell membrane receptors. This leads to an affinity at least 26-fold higher than the intrinsic affinity of the original aptamer probes. As determined by flow cytometric measurements, an enhancement in fluorescence signal in excess of 300-fold is obtained for the NR-aptamer-labeled cells compared with those labeled by individual aptamer probes. Therefore, the molecular assembly of aptamers clearly shows potential applications for the elucidation of cells with low density of binding sites, or with relatively weak binding probes, and can thus greatly improve our ability to perform cellular imaging and targeting. This is an excellent example of using nanomaterials to develop advanced molecular binders with greatly improved properties for cellular studies.     

Measurement of aptamer-protein interactions with back-scattering interferometry

We report the quantitative measurement of aptamer-protein interactions using backscattering interferometry (BSI) and show that BSI can determine when distinct binding regions are accessed. As a model system, we utilized two DNA aptamers (Tasset and Bock) that bind to distinct sites of a target protein (human α-thrombin). This is the first time BSI has been used to study a multivalent system in free solution wherein more than one ligand binds to a single target. We measured aptamer equilibrum dissociation constants (K(d)) of 3.84 nM (Tasset-thrombin) and 5.96 nM (Bock-thrombin), in close agreement with the literature. Unexpectedly, we observed allosteric effects such that the binding of the first aptamer resulted in a significant change in the binding affinity of the second aptamer. For example, the K(d) of Bock aptamer binding to preformed Tasset-thrombin complexes was 7-fold lower (indicating higher affinity) compared to binding to thrombin alone. Preliminary modeling efforts suggest evidence for allosteric linkage between the two exosites.     

Selection of smart aptamers by methods of kinetic capillary electrophoresis

We coin the term "smart aptamers" -- aptamers with predefined binding parameters (k(on), k(off), Kd) of aptamer-target interaction. Aptamers, in general, are oligonucleotides, which are capable of binding target molecules with high affinity and selectivity. They are considered as potential therapeutic targets and also thought to rival antibodies in immunoassay-like analyses. Aptamers are selected from combinatorial libraries of oligonucleotides by affinity methods. Until now, technological limitations have precluded the development of smart aptamers. Here, we report on two kinetic capillary electrophoresis techniques applicable to the selection of smart aptamers. Equilibrium capillary electrophoresis of equilibrium mixtures was used to develop aptamers with predefined equilibrium dissociation constants (Kd), while nonequilibrium capillary electrophoresis of equilibrium mixtures facilitated selection of aptamers with different dissociation rate constants (k(off)). Selections were made for MutS protein, for which aptamers have never been previously developed. Both theoretical and practical aspects of smart aptamer development are presented, and the advantages of this new type of affinity probes are described.     

Selection of aptamers against live bacterial cells

Single-stranded DNA or RNA aptamer molecules have usually been selected against purified target molecules. To eliminate the need of purifying target molecules on the cell surface, we have developed a selection technique using live bacterial cells in suspension as targets, to select for ssDNA aptamers specific to cell surface molecules. Lactobacillus acidophilus cells were chosen to demonstrate proof of principle based on their high abundance of surface molecules (potential targets). Aptamer pools obtained after 6-8 rounds of selection demonstrated high affinity for and selective binding with L. acidophilus cells when tested via flow cytometry, microscopy, and fluorescence measurements. Out of 27 aptamers that were cloned and sequenced, one sequence, hemag1P, was found to bind to L. acidophilus much more strongly and specifically than other cells tested. This aptamer was predicted to have a tight hairpin secondary structure. On average, an estimated 164 +/- 47 aptamer molecules were bound to a target cell with an apparent K d of 13 +/- 3 nM. A likely putative molecular target of hemag1P is the S-layer protein on the cell surface.     

Surface-immobilized peptide aptamers as probe molecules for protein detection

We demonstrate the use of surface-immobilized, oriented peptide aptamers for the detection of specific target proteins from complex biological solutions. These peptide aptamers are target-specific peptides expressed within a protein scaffold engineered from the human protease inhibitor stefin A. The scaffold provides stability to the inserted peptides and increases their binding affinity owing to the resulting three-dimensional constraints. A unique cysteine residue was introduced into the protein scaffold to allow orientation-specific surface immobilization of the peptide aptamer and to ensure exposure of the binding site to the target solution. Using dual-polarization interferometry, we demonstrate a strong relationship between binding affinity and aptamer orientation and determine the affinity constant KD for the interaction between an oriented peptide aptamer ST(cys+)_(pep9) and the target protein CDK2. Further, we demonstrate the high selectivity of the peptide aptamer STM_(pep9) by exposing surface-immobilized ST(cys+)_(pep9) to a complex biological solution containing small concentrations of the target protein CDK2.     

Ultrasensitive detection of protein using an aptamer-based exonuclease protection assay

Currently, methods for protein detection are not as sensitive and specific as methods for detection of specific nucleic acid sequences. Here, we present an analogous technique for detection of proteins using aptamers as ligands for target binding. We have named this method the aptamer-based exonuclease protection assay. We applied a special oligonucleotide probe containing a thrombin aptamer, which has the capacity to recognize thrombin with high affinity and specificity. The aptamer probe is a 22-base-long single-strand oligonucleotide with the thrombin aptamer sequence at the 3'-terminus and 7 additional nucleotides at the 5'-terminus, which is able to bind thrombin with high affinity and specificity. In the exonuclease protection assay, thrombin binds the aptamer and thereby protects it from degradation by exonuclease I, whereas any unbound aptamer probe is degraded by exonuclease I. Subsequently, the aptamer probes that were protected from exonuclease I by thrombin act as linkers to join two free connectors, which contain sequences matching the probe. The joined products, which reflect the identity and amount of the target protein, are amplified by PCR. The exonuclease protection assay is extremely sensitive, since it is based on PCR amplification. This method can detect as few as several hundred molecules of target protein without using washes or separations. In addition, this new method for protein detection is simple and inherits all the advantages of aptamers. The mechanism, moreover, may be generalized and used for other forms of protein analysis.     

RNA‐Capturing Microsphere Particles (R‐CAMPs) for Optimization of Functional Aptamers

RNA aptamers are useful building blocks for constructing functional nucleic acid‐based nanoarchitectures. The abilities of aptamers to recognize specific ligands have also been utilized for various biotechnological applications. Solution conditions, which can differ depending on the application, impact the affinity of the aptamers, and thus it is important to optimize the aptamers for the solution conditions to be employed. To simplify the aptamer optimization process, an efficient method that enables re‐selection of an aptamer from a partially randomized library is developed. The process relies on RNA‐capturing microsphere particles (R‐CAMPs): each particle displays different clones of identical DNA and RNA sequences. Using a fluorescence‐activated cell sorter, the R‐CAMPs that are linked to functional aptamers are sorted. It is demonstrated that after a single round of reselection, several functional aptamers, including the wild‐type, are selected from a library of 16 384 sequences. The selection using R‐CAMPs is further performed under the solution containing high concentration of ethylene glycol, suggesting applicability in various conditions to optimize an aptamer for a particular application. As any type of RNA clone can be displayed on the microspheres, the technology demonstrated here will be useful for the selection of RNAs based on diverse functions.     

Systematic investigation of optimal aptamer immobilization for protein-microarray applications

Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to a wide range of target molecules with high affinity and specificity. As nucleic acids, aptamers can undergo denaturation, but the process is reversible. As a result of this stability and the possibility of automated selection of aptamers, these oligonucleotides are highly promising capture molecules in microarray formats. In this study, his-tagged proteins and an aptamer directed against the his-tag were chosen as a model system. Different factors affect the activity of aptamers immobilized on a solid support like a microarray surface. The orientation of the immobilized aptamer plays an important role in correct aptamer folding and, thus, in effective binding of the corresponding target. Other important parameters identified in this work are the microarrays' surface charge as well as the length of the spacer between aptamer and solid support. These parameters were investigated systematically, resulting in the development of an aptamer-based microarray for detection of his-tagged proteins. The general applicability of the developed immobilization strategy was demonstrated by utilization of three different aptamers.     

Study of the effect of metal ion on the specific interaction between protein and aptamer by atomic force microscopy

We have studied the effect of metal ions on the specific interaction between a protein, immunoglobulin E (IgE), and its 37-nt DNA aptamer with atomic force microscopy (AFM). Protein aptamers are a new class of synthetic single-stranded DNA/RNA oligonucleotide generated from in vitro selection to selectively bind with target proteins. The IgE aptamers have been developed and are expected to be promising reagents in IgE detection and new anti-allergic drug development. It is known that the presence of metal ions in the buffer usually has a strong effect on the affinity of single-stranded DNA for protein. In this work, the effect of two representative monovalent ion and divalent ion on the binding of IgE and the aptamer has been studied at the single-molecule level. The results from the AFM force measurements show that the metal ions not only reduce the single-molecular rupture force but also reduce the number of bonds formed between IgE and the aptamer.     

DNA aptamers binding to multiple prevalent M-types of Streptococcus pyogenes

This paper describes the selection of high affinity DNA aptamers binding to multiple M-types of the pathogenic species Streptococcus pyogenes (Group A Streptococcus or GAS). Unlike common aptamer selection techniques that use purified molecules of a monoclonal cell population as targets, this work has achieved the selection of aptamers against the various M-types of S. pyogenes. Cell mixtures containing equal numbers of the 10 most prevalent S. pyogenes M-types were incubated with 80-nucleotide DNA libraries, centrifuged, and washed to separate cell-bound from unbound DNA sequences. The DNA bound to the cells was amplified using the polymerase chain reaction, and the amplicons were tested for their binding to the target cells. The amplicons were also used as new DNA libraries for subsequent rounds of selection. Cloning, sequencing, and subsequent analysis of selected aptamers showed that they bind preferentially to GAS over other common and related bacteria. Resultant DNA aptamers showed strong and preferential binding to GAS, including the 10 most prevalent GAS M-types and another 10 minor M-types tested. Estimated K(d) values were in the range of 4 to 86 nM. Two aptamers, 20A24P and 15A3P (with estimated binding dissociation constants of 9 and 10 nM, respectively), are particularly promising. These aptamers could potentially be used to improve the detection of GAS, a pathogen that is the causative agent of many infectious diseases, most notably strep throat.     

Aptamer-derived nucleic acid oligos: applications to develop nucleic acid chips to analyze proteins and small ligands

The specificity and affinity of aptamers for their cognate ligands are comparable to those of antibodies for antigens. To use aptamers effectively in high-throughput assays in a microarray format, to analyze various analytes, we developed a strategy in which the aptamer was split into two nonfunctional units and allowed to reassemble into the functional aptamer by the cognate ligand. We have named this method "analyte-dependent oligonucleotide modulation assay" (ADONMA). As proof-of-principle, we used oligonucleotides derived from the aptamer RNA against HIV-1 Tat and demonstrated, with both titer plates and plastic slide chips, that specifically in the presence of Tat or its peptide, the two oligos reconstituted the core binding regions of Tat. Thus, these results suggest that ADNOMA has the potential for use in nucleic acid microarrays for detecting various ligands.     

Aptamer-functionalized microgel particles for protein detection

Highly sensitive and multiplexed detection of clinically relevant proteins in biologically complex samples is crucial for the advancement of clinical proteomics. In recent years, aptamers have emerged as useful tools for protein analysis due to their specificity and affinity for protein targets as well as their compatibility with particle-based detection systems. In this study, we demonstrate the highly sensitive detection of human α-thrombin on encoded hydrogel microparticles functionalized with an aptamer capture sequence. We use static imaging and microfluidic flow-through analysis techniques to evaluate the detection capabilities of the microgels in sandwich-assay formats that utilize both aptamers and antibodies for the reporting of target-binding events. Buffers and reagent concentrations were optimized to provide maximum reaction efficiency while still maintaining an assay with a simple workflow that can be easily adapted to the multiplexed detection of other clinically relevant proteins. The three-dimensional, nonfouling hydrogel immobilization scaffold used in this work provides three logs of dynamic range, with a limit of detection of 4 pM using a single aptamer capture species and without the need for spacers or signal amplification.     

Selection of DNA aptamers that recognize α-synuclein oligomers using a competitive screening method

α-Synuclein (α-syn) oligomers are considered major molecules responsible for the onset of Parkinson's disease and dementia with Lewy bodies. α-Syn oligomers thus serve as an important target for the development of drugs and diagnostic tests for neurodegenerative diseases. In this paper we report on the identification of DNA aptamers that bind to soluble α-syn oligomers. A competitive screening method based on aptamer blotting was used for the selection of α-syn oligomer-specific aptamers. This approach resulted in the identification of eight aptamers that specifically bind to α-syn oligomers among α-syn monomers, oligomers, and fibrils. Interestingly, the aptamers also bound to amyloid β oligomers, which are strongly associated with the development of Alzheimer's disease. The results of this study support the hypothesis that amyloid oligomers share a common structure. Oligomer-binding aptamers may serve as powerful analytical tools for the design and development of drugs and diagnostic tests for neurodegenerative diseases.     

Displacement enzyme linked aptamer assay

Immense effort has been placed on the realization of immunoassays exploiting displacement of a suboptimum target, due to the ease of use and applicability to immunochromatographic strips and immunosensors. Most of the efforts reported to date focus on the use of a suboptimal target that is displaceable by the target toward which the antibody has higher affinity. Limited success has been achieved due to difficulty in obtaining suboptimal targets to which the antibody has enough affinity to bind while at the same time having lower levels of affinity in comparison to the target to facilitate displacement. Aptamers are synthetic oligonucleotides specifically selected to bind a certain target. Thanks to their high affinity and sensitivity, aptamers appear as alternative candidates to antibodies for analytical devices and several enzyme-linked aptamer assays and aptasensors have been reported. Aptamers, in contrast to antibodies, require the formation of a three-dimensional structure for target binding and can thus be anticipated to have a much higher affinity for binding its target rather than a modified form of the target (e.g., enzyme-labeled target). This phenomenon can be exploited for the development of a displacement assay, using enzyme-labeled target as a suboptimal displaceable molecule. Here, we report the first demonstration of the exploitation of an aptamer in an extremely rapid and highly sensitive displacement assay. Surface plasmon resonance studies demonstrated the thrombin-binding aptamer to have a lower affinity for enzyme-labeled thrombin than unmodified thrombin, with respective K(D) of 1.1 x 10(-8) and 2.9 x 10(-9) M. The assay is extremely rapid, requiring only 10 min for completion, and exhibits a detection limit lower than that obtainable with competitive enzyme-linked aptamer assays and comparable to that of hybrid aptamer-antibody assays. Optimal storage conditions for precoated microtiter plates (consisting of coated aptamer and captured labeled target) were elucidated, and the results demonstrated their amenability to long-term storage, facilitating commercially viable displacement enzyme-linked aptamer assays that simply require sample addition, with a total assay time, including color development, of 30 min.     

Reusable electrochemical sensing platform for highly sensitive detection of small molecules based on structure-switching signaling aptamers

Aptamers are nucleic acids that have high affinity and selectivity for their target molecules. A target may induce the structure switching from a DNA/DNA duplex to a DNA/target complex. In the present study, a reusable electrochemical sensing platform based on structure-switching signaling aptamers for highly sensitive detection of small molecules is developed using adenosine as a model analyte. A gold electrode is first modified with polytyramine and gold nanoparticles. Then, thiolated capture probe is assembled onto the modified electrode surface via sulfur-gold affinity. Ferrocene (Fc)-labeled aptamer probe, which is designed to hybridize with capture DNA sequence and specifically recognize adenosine, is immobilized on the electrode surface by hybridization reaction. The introduction of adenosine triggers structure switching of the aptamer. As a result, Fc-labeled aptamer probe is forced to dissociate from the sensing interface, resulting in a decrease in redox current. The decrement of peak current is proportional to the amount of adenosine. The present sensing system could provide both a wide linear dynamic range and a low detection limit. In addition, high selectivity, good reproducibility, stability, and reusability are achieved. The recovery test demonstrates the feasibility of the designed sensing system for an adenosine assay.     

Systematic Aptamer-Gold Nanoparticle Colorimetry for Protein Detection: Thrombin

Gold nanoparticle colorimetry assay using aptamers is a low cost and a highly effective means for detecting a wide range of biomolecular targets. In this work, this technique is used to detect the protein thrombin as a model system for understanding the relationship between the aptamer-target binding properties and the optical colorimetric response, as well as to gain insight on the secondary structures of the aptamers. The two known aptamers for thrombin, the 15-mer Bock and the 29-mer Tasset aptamer were conjugated to gold nanoparticles to form complexes that bind to thrombin upon contact. The Bock aptamer causes the aggregation of the nanoparticles and the concomitant reduction of the plasmon resonance peak, whereas the 29-mer Tasset aptamer, despite higher affinity, does not cause a spectral change. The data is understood on the basis of the difference in the number of binding sites available on thrombin for the respective aptamers. Additional results on single base substitutions suggest that the G-quadruplex secondary structure in the Bock aptamer is intermolecular and comprises of at least two interacting aptamer molecules. An estimate of the dissociation constant, derived from thrombin titration, is comparable to values reported in the literature.     

Aptamer‐Engineered Natural Killer Cells for Cell‐Specific Adaptive Immunotherapy

Natural killer (NK) cells are a key component of the innate immune system as they can attack cancer cells without prior sensitization. However, due to lack of cell‐specific receptors, NK cells are not innately able to perform targeted cancer immunotherapy. Aptamers are short single‐stranded oligonucleotides that specifically recognize their targets with high affinity in a similar manner to antibodies. To render NK cells with target‐specificity, synthetic CD30‐specific aptamers are anchored on cell surfaces to produce aptamer‐engineered NK cells (ApEn‐NK) without genetic alteration or cell damage. Under surface‐anchored aptamer guidance, ApEn‐NK specifically bind to CD30‐expressing lymphoma cells but do not react to off‐target cells. The resulting specific cell binding of ApEn‐NK triggers higher apoptosis/death rates of lymphoma cells compared to parental NK cells. Additionally, experiments with primary human NK cells demonstrate the potential of ApEn‐NK to specifically target and kill lymphoma cells, thus presenting a potential new approach for targeted immunotherapy by NK cells.     

Aptasensor development: elucidation of critical parameters for optimal aptamer performance

Aptamers are synthetic oligonucleotides specifically selected to bind a certain target. Thanks to their high affinity and sensitivity, aptamers appear as alternative candidates to antibodies for analytical devices and several assays have been reported. However, and contrary to what happens with DNA probes, the aptamers' ability to bind their targets depends on folding and 3-D structure, which may be affected by the incubation conditions and buffer composition. In this report, a systematic evaluation of the parameters with potential effect on the ELAA (Enzyme Linked Aptamer Assay) performance has been carried out. Additionally, diverse ELAA and mixed ELISA/ELAA formats exploiting the thrombin-binding aptamer have been optimized and their efficiencies compared. ELAA results have been confirmed using nuclear magnetic resonance, electrophoresis, and surface plasmon resonance. Our results indicate that parameters such as immobilization strategy, incubation time/temperature, and buffer composition should be optimized for each aptamer as they affect folding and, thus, binding efficiency. Among the studied assays, the mixed ELISA/ELAA sandwich formats showed the lowest limit of detection observed (<1 nM thrombin), while a competition ELAA appeared as the best assay in terms of high sensitivity (1.8 nM) and short assay time (1 h, 30 min). The elucidation of optimal parameters for assay performance reported here clearly indicates that aptamers are unique structures. Formation of the 3-D structures required for target binding is influenced by variable parameters, and unlike DNA/antibody based assays, there are no general recommendations, with each assay requiring individual optimization of parameters.