Multiple Aptamers Research Articles

Tuning Biosensor Cross-Reactivity Using Aptamer Mixtures.

It is challenging to tune the response of biosensors to a set of ligands, for example, cross-reactivity to a given target family while maintaining high specificity against interferents, due to the lack of suitable bioreceptors. We present a novel approach for controlling the cross-reactivity of biosensors by employing defined mixtures of aptamers that have differing binding properties. As a demonstration, we develop assays for the specific detection of a family of illicit designer drugs, the synthetic cathinones, with customized responses to each target ligand and interferent. We first use a colorimetric dye-displacement assay to show that the binding spectra of dual-aptamer mixtures can be tuned by altering the molar ratio of these bioreceptors. Optimized assays achieve broad detection of synthetic cathinones with minimal response toward interferents and generally demonstrate better sensing performance than assays utilizing either aptamer alone. The generality of this strategy is demonstrated with a dual-aptamer electrochemical sensor. Our approach enables customization of biosensor responsiveness to an extent that has yet to be achieved through any previously reported aptamer engineering techniques such as sequence mutation or truncation. Since multiple aptamers for the designated target family can routinely be identified via high-throughput sequencing, we believe our strategy offers a generally applicable method for generating near-ideal aptamer biosensors for various analytical applications, including medical diagnostics, environmental monitoring, and drug detection.

Screening of multiple hemoprotein-specific aptamers and their applications for the binding, quantification, and extraction of hemoproteins in a microfluidic system.

The blood hemoproteins, albumin, γ-globulin, and fibrinogen, serve as biomarkers for a variety of human diseases, including kidney and hepatorenal syndromes. Therefore, there is a need to quickly and accurately measure their concentrations in blood. Herein, nucleic acid aptamers demonstrating high affinity and specificity toward these hemoproteins were selected via systematic evolution of ligands by exponential enrichment, and their ability to capture their protein targets was assessed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a tetramethyl benzidine assay. The limits of detection for the hemoproteins were all around 10-3 μM, and dissociation constant values of 131, 639, and 29nM were obtained; capture rates were measured to be 66%, 71%, and 61%, which is likely to be suitable for clinical diagnostics. Furthermore, a multi-layer microfluidic disk system featuring hemoprotein-specific aptamers for depleting hemoproteins was demonstrated. It could be a promising approach to use aptamers to replace conventional antibodies.

AptCompare: optimized de novo motif discovery of RNA aptamers via HTS-SELEX.

High-throughput sequencing can enhance the analysis of aptamer libraries generated by the Systematic Evolution of Ligands by EXponential enrichment. Robust analysis of the resulting sequenced rounds is best implemented by determining a ranked consensus of reads following the processing by multiple aptamer detection algorithms. While several such approaches have been developed to this end, their installation and implementation is problematic. We developed AptCompare, a cross-platform program that combines six of the most widely used analytical approaches for the identification of RNA aptamer motifs and uses a simple weighted ranking to order the candidate aptamers, all driven within the same GUI-enabled environment. We demonstrate AptCompare's performance by identifying the top-ranked candidate aptamers from a previously published selection experiment in our laboratory, with follow-up bench assays demonstrating good correspondence between the sequences' rankings and their binding affinities. The source code and pre-built virtual machine images are freely available at https://bitbucket.org/shiehk/aptcompare. Supplementary data are available at Bioinformatics online.

Capture and selective release of multiple types of circulating tumor cells using smart DNAzyme probes.

The effective capture, release and reanalysis of circulating tumor cells (CTCs) are of great significance to acquire tumor information and promote the progress of tumor therapy. Particularly, the selective release of multiple types of CTCs is critical to further study; however, it is still a great challenge. To meet this challenge, we designed a smart DNAzyme probe-based platform. By combining multiple targeting aptamers and multiple metal ion responsive DNAzymes, efficient capture and selective release of multiple types CTCs were realized. Sgc8c aptamer integrated Cu2+-dependent DNAzyme and TD05 aptamer integrated Mg2+-dependent DNAzyme can capture CCRF-CEM cells and Ramos cells respectively on the substrate. With the addition of Cu2+ or Mg2+, CCRF-CEM cells or Ramos cells will be released from the substrate with specific selectivity. Furthermore, our platform has been successfully demonstrated in the whole blood sample. Therefore, our capture/release platform will benefit research on the molecular analysis of CTCs after release and has great potential for cancer diagnosis and individualized treatment.

Silicon-Dioxide-Specific Peptides for Biological Nanofabrication: Selecting Aptamers for Target-Specific Ferritin Supramolecule Delivery

Aptamers with 12 amino acid peptides that specifically bind to silicon dioxide (SiO2) were obtained through the phage-display method. A quartz crystal microbalance (QCM) measurement showed that the peptides bound to the SiO2 surface quickly, and that the lowest dissociation constant (Kd) was 16 micromolars (mM). The recombinant ferritins, cage-shaped protein mutants that had the aptamers on their outer surface, showed a much higher binding affinity to the SiO2 surface, and the Kd reached as low as 2.3 nm. The results indicated that multiple aptamers coordinately bound to the SiO2 surface. It was demonstrated that the aptamers on the outer surface had the ability to selectively deliver the recombinant ferritins with a nanoparticle (NP) to the SiO2 surface. The NP array made by the recombinant ferritin on the SiO2 substrate could be used as a nanoetching mask. This technique for making an NP array through ferritins is being applied to other cage-shaped proteins.

Development of a sensitive DMD-based 2D SPR sensor array using single-point detection strategy for multiple aptamer screening

In this paper, we report a novel single-point detection strategy for 2D solid-state angular interrogated SPR (surface plasmon resonance) sensor array for multiple aptamer screening. The scheme operates with two DMDs (digital micro-mirror devices), which suffices a single-point detector for high-throughput 2D SPR measurements without sacrificing the sensitivity. After having verified the biosensing capability of the system, in terms of sensitivity and repeatability, using standard BSA-anti-BSA antibody interaction, we further conducted experiments to perform rapid screening of aptamer candidates for binding with tumor necrosis factor-α (TNF-α). The selected anti-TNF-α aptamers were subsequently evaluated as an in vitro cell protection agent against TNF-α-induced cell death. Our experiments confirm that the anti-TNF-α aptamer screening approach offers high potential as an alternative to that based on antibodies for diagnostic and therapeutic applications.

Construction of a Multiple-Aptamer-Based DNA Logic Device on Live Cell Membranes via Associative Toehold Activation for Accurate Cancer Cell Identification.

The ability to accurately identify and isolate cells is the cornerstone of precise disease diagnosis and therapies. A single-step cell identification method based on logic analysis of multiple surface markers will have unique advantages because of its accuracy and efficacy. Herein, using multiple DNA aptamers for cancer biomarker recognition and associative toehold activation for signal integration and amplification as two molecular keys, we have successfully operated a cell-surface device that can perform AND Boolean logic analysis of multiple biomarkers and precisely label the target cell subtype in large populations of similar cells via the presence or absence of different biomarkers. Our approach can achieve single-step cancer cell identification and isolation with excellent sensitivity and accuracy and thus will have broad applications in biological science, biomedical engineering, and personalized medicine.

Traceless aptamer-mediated isolation of CD8+ T cells for chimeric antigen receptor T-cell therapy

Chimeric antigen receptor (CAR) T-cell therapies using defined product compositions require high-purity T-cell isolation systems that, unlike immunomagnetic positive enrichment, are inexpensive and leave no trace on the final cell product. Here, we show that DNA aptamers, generated with a modified cell-SELEX (systematic evolution of ligands by exponential enrichment) procedure to display low nanomolar affinity for the T-cell marker CD8, enable the traceless isolation of pure CD8+ T cells at low cost and high yield. Captured CD8+ T cells are released label-free by complementary oligonucleotides that undergo toehold-mediated strand displacement with the aptamer. We also show that CAR-T cells manufactured from these cells are comparable to antibody-isolated CAR-T cells in proliferation, phenotype, effector function and antitumor activity in a mouse model of B-cell lymphoma. By employing multiple aptamers and the corresponding complementary oligonucleotides, aptamer-mediated cell selection could enable the fully synthetic, sequential and traceless isolation of desired lymphocyte subsets from a single system.

Integrating Ligand-Receptor Interactions and In Vitro Evolution for Streamlined Discovery of Artificial Nucleic Acid Ligands

To discover DNA ligands against a predetermined receptor protein complex, we introduce a comprehensive version of ligand-guided selection (LIGS). LIGS is, itself, a variant of systematic evolution of ligands by exponential enrichment (SELEX). Herein, we have optimized LIGS to identify higher affinity aptamers with high specificity. In addition, we demonstrate the expandability of LIGS by performing specific aptamer elution at 25°C, utilizing multiple monoclonal antibodies (mAbs) against cultured cells and primary cells obtained from human donors expressing the same receptor. Eluted LIGS libraries obtained through Illumina high-throughput (HT) DNA sequencing were analyzed by bioinformatics tools to discover five DNA aptamers with apparent affinities ranging from 3.06 ± 0.485 nM to 325 ± 62.7 nM against the target, T cell receptor-cluster of differentiation epsilon (TCR-CD3ε) expressed on human T cells. The specificity of the aptamers was validated utilizing multiple strategies, including competitive binding analysis and a double-knockout Jurkat cell line generated by CRISPR technology. The cross-competition experiments using labeled and unlabeled aptamers revealed that all five aptamers compete for the same binding site. Collectively, the data in this report introduce a modified LIGS strategy as a universal platform to identify highly specific multiple aptamers toward multi-component receptor proteins in their native state without changing the cell-surface landscape.

Terminal Deoxynucleotidyl Transferase-Catalyzed Preparation of pH-Responsive DNA Nanocarriers for Tumor-Targeted Drug Delivery and Therapy.

Developing a highly efficient carrier for tumor-targeted delivery and site-specific release of anticancer drugs is a good way to overcome the side effects of traditional cancer chemotherapy. Benefiting from the nontoxic and biocompatible characteristics, DNA-based drug carriers have attracted increasing attention. Herein, we reported a novel and readily manipulated strategy to construct spherical DNA nanocarriers. In this strategy, terminal deoxynucleotidyl transferase (TdT)-catalyzed DNA extension reaction is used to prepare a thick DNA layer on a gold nanoparticle (AuNP) surface by extending long poly(C) sequences from DNA primers immobilized on AuNPs. The poly(C) extension products can then hybridize with G-rich oligonucleotides to give CG-rich DNA duplexes (for loading anticancer drug doxorubicin, Dox) and multiple AS1411 aptamers. Via synergic recognition of multiple aptamer units to nucleolin proteins, biomarker of malignant tumors, Dox-loaded DNA carrier can be efficiently internalized in cancer cells and achieve burst release of drugs in acidic organelles because of i-motif formation-induced DNA duplex destruction. An as-prepared pH-responsive drug carrier was demonstrated to be promising for highly efficient delivery of Dox and selective killing of cancer cells in both in vitro and in vivo experiments, thus showing a huge potential in anticancer therapy.

Screening of DNA Signaling Aptamer from Multiple Candidates Obtained from SELEX with Next-generation Sequencing.

Here, we demonstrated a strategy for developing signaling aptamers, based on screening of signaling aptamers from multiple aptamer candidates obtained by SELEX with next generation sequencing. Among aptamer candidates labelled by 6-carboxyfluorescein and quencher at both end termini, there is the possibility of discovering a potent signaling aptamer. In this study, we discovered DNA signaling aptamers against VEGFR-1. This strategy has the potential for signaling aptamer discovery without the extremely laborious task of optimization of oligodeoxynucleotide modifications.

Single-round isolation of diverse RNA aptamers from a random sequence pool.

Aptamers are oligonucleotide ligands with specific binding affinity to target molecules. Generally, RNA aptamers are selected from an RNA pool with random sequences, using the technique termed SELEX, in which the target-binding RNA molecules are repeatedly isolated and exponentially amplified. Despite several advantages, SELEX often produces uncertain results during the iterative amplifications of the rare target-binding RNA molecules. Here, we develop a non-repeated, primer-less and target immobilization-free isolation method for generating RNA aptamers, which is robust to experimental noise. Uniquely, this method focuses on finding and removal of non-aptamer sequences from the RNA pool by RNase digestion leaving target-bound aptamer molecules, and thus is independent of aptamer types. The undigested RNA sequences remaining are so few in number that they must be mixed with a large excess of a known sequence for further manipulations and this sequence is then removed by restriction digestion followed by high-throughput sequencing analysis to identify aptamers. Using this method, we generated multiple RNA aptamers targeting α-thrombin and TGFβ1 proteins, independently. This method potentially generates thousands of sequences as aptamer candidates, which may enable us to predict a common average sequence or structural property of these aptamers that is different from input RNA.

Enhanced Targeted Gene Transduction: AAV2 Vectors Conjugated to Multiple Aptamers via Reducible Disulfide Linkages.

Enhanced targeted gene transduction by AAV2 vectors is achieved by linking the vector to multiple sgc8 aptamers, which are selective for cell membrane protein PTK7. Aptamer molecules are conjugated to multiple sites on a DNA dendrimer (G-sgc8), which is then linked to AAV2 via a dithiobis(succinimidyl propionate) cross-linker containing a disulfide group, which can facilitate the release of AAV2 vectors by reaction with the reduced form of intracellular glutathione. The G-sgc8-AAV2 vectors showed a 21-fold enhancement in binding affinity and an enhanced ability to protect sgc8 aptamers against nuclease degradation to cells expressing PTK7 compared to single aptamer-AAV2 conjugates. The transduction efficiency was tested by loading AAV2 with the gene for green fluorescent protein. Therefore, this modified recombinant vector is an attractive and promising tool for targeted biomedical applications.

Enumeration of circulating tumor cells and investigation of cellular responses using aptamer-immobilized AlGaN/GaN high electron mobility transistor sensor array

This study reports the fabrication and characterization of electrical double layer gated AlGaN/GaN High electron mobility (HEMT) biosensor array to capture, detect and count circulating tumor cells (CTCs) of colorectal cancer (CRC). GaN HEMT chips were assembled into a sensor array on a thermo-curable polymer substrate in a simple and robust packaging process. The array had multiple aptamer immobilized sensing sites and was highly sensitive and selective with up to single cell resolution. The present method can detect cells in their native electrolyte composition, in a very short time with extremely low cost compared to the conventional circulating tumor cell assays. The effect of cellular binding in different test environments was further studied and reported. The electrical response of the sensor was discovered to be relevant to the transmembrane potential of the cell. This whole cell sensor array has the potential to be used as a point-of-care diagnostic tool in varied applications such as detection of rare cells, pathogens and studying the dynamics of cellular interactions.

Multivalent Aptamer-RNA Conjugates for Simple and Efficient Delivery of Doxorubicin/siRNA into Multidrug-Resistant Cells.

Multivalent aptamer-siRNA conjugates containing multiple mucin-1 aptamers and BCL2-specific siRNA are synthesized, and doxorubicin, an anthracycline anticancer drug, is loaded into these conjugates through intercalation with nucleic acids. These doxorubicin-incorporated multivalent aptamer-siRNA conjugates are transfected to mucin-1 overexpressing MCF-7 breast cancer cells and their multidrug-resistant cell lines. Doxorubicin-incorporated multivalent aptamer-siRNA conjugates exert promising anticancer effects, such as activation of caspase-3/7 and decrease of cell viability, on multidrug-resistant cancer cells because of their high intracellular uptake efficiency. Thus, this delivery system is an efficient tool for combination oncotherapy with chemotherapeutics and nucleic acid drugs to overcome multidrug resistance.

High-Throughput Discovery of Aptamers for Sandwich Assays.

Sandwich assays are among the most powerful tools in molecular detection. These assays use "pairs" of affinity reagents so that the detection signal is generated only when both reagents bind simultaneously to different sites on the target molecule, enabling highly sensitive and specific measurements in complex samples. Thus, the capability to efficiently screen affinity reagent pairs at a high throughput is critical. In this work, we describe an experimental strategy for screening "aptamer pairs" at a throughput of 106 aptamer pairs per hour-which is many orders of magnitude higher than the current state of the art. The key step in our process is the conversion of solution-phase aptamers into "aptamer particles" such that we can directly measure the simultaneous binding of multiple aptamers to a target protein based on fluorescence signals and sort individual particles harboring aptamer pairs via the fluorescence-activated cell-sorter instrument. As proof of principle, we successfully isolated a high-quality DNA aptamer pair for plasminogen activator inhibitor 1 (PAI-1). Within only two rounds of screening, we discovered DNA aptamer pairs with low-nanomolar sensitivity in dilute serum and excellent specificity with minimal off-target binding even to closely related proteins such as PAI-2.

744. Optimizing Conditions for Aptamer Folding Using a High-Throughput Aptamer Fluorescence Binding and Internalization (AFBI) Assay

Aptamers are small structured RNA or DNA oligonucleotides that bind target molecules with high avidity and affinity. Aptamer selection begins with a hugely complex library of aptamer sequences that are enriched for a specific target using the SELEX process, Systematic Evolution of Ligands by EXponentional enrichment. An important consideration in the SELEX process is the protocol used to fold aptamers into their active conformations. The folding conditions include multiple variables, such as temperature, buffer components, incubation time and aptamer concentration. Aptamer folding protocols vary widely across the aptamer field, and most published folding conditions primarily describe only temperature and folding time. To understand how variations in folding conditions impact aptamer function, we developed a novel high-throughput assay to interrogate the optimal folding conditions of several published aptamers. The Aptamer Fluorescence Binding and Internalization (AFBI) assay is a cell-based platform that uses a 96-well plate format to rapidly and efficiently screen multiple fluorescent-labelled aptamers against hundreds of conditions. The AFBI assay can be applied to rapidly determine aptamer binding constants (Kd) on cells, time course of aptamer internalization and cross reactivity of aptamers against different cell types.Using the AFBI assay, we screened several different folding parameters against published aptamers. We found that the buffer components contributed significantly more to an aptamer's function than any other examined factor. The concentration of an aptamer during folding was important for some aptamers but not others, including aptamers that originated from the same selection. Most surprising was that most temperature protocols had little impact on aptamer function after folding, the exception being that high temperature (95°C) often attenuated aptamer function. In summary, our data using the AFBI assay revealed that aptamer folding is more dependent on buffer components than the temperature protocol. Furthermore, aptamers derived from the same selection may have different optimal folding conditions. These data demonstrate that optimal aptamer folding protocols need to be more carefully interrogated on a per aptamer basis and reported in detail to allow for efficacious and reproducible results.

Design of Potent and Controllable Anticoagulants Using DNA Aptamers and Nanostructures.

The regulation of thrombin activity offers an opportunity to regulate blood clotting because of the central role played by this molecule in the coagulation cascade. Thrombin-binding DNA aptamers have been used to inhibit thrombin activity. In the past, to address the low efficacy reported for these aptamers during clinical trials, multiple aptamers have been linked using DNA nanostructures. Here, we modify that strategy by linking multiple copies of various thrombin-binding aptamers using DNA weave tiles. The resulting constructs have very high anticoagulant activity in functional assays owing to their improved cooperative binding affinity to thrombin due to optimized spacing, orientation, and the high local concentration of aptamers. We also report the results of molecular dynamics simulations to gain insight into the solution conformations of the tiles. Moreover, by using DNA strand displacement, we were able to turn the coagulation cascade off and on as desired, thereby enabling significantly better control over blood coagulation.

Enhanced and Differential Capture of Circulating Tumor Cells from Lung Cancer Patients by Microfluidic Assays Using Aptamer Cocktail.

Collecting circulating tumor cells (CTCs) shed from solid tumor through a minimally invasive approach provides an opportunity to solve a long-standing oncology problem, the real-time monitoring of tumor state and analysis of tumor heterogeneity. However, efficient capture and detection of CTCs with diverse phenotypes is still challenging. In this work, a microfluidic assay is developed using the rationally-designed aptamer cocktails with synergistic effect. Enhanced and differential capture of CTCs for nonsmall cell lung cancer (NSCLC) patients is achieved. It is also demonstrated that the overall consideration of CTC counts obtained by multiple aptamer combinations can provide more comprehensive information in treatment monitoring.

A Targeted "Capture" and "Removal" Scavenger toward Multiple Pollutants for Water Remediation based on Molecular Recognition.

For the water remediation techniques based on adsorption, the long‐standing contradictories between selectivity and multiple adsorbability, as well as between affinity and recyclability, have put it on weak defense amid more and more severe environment crisis. Here, a pollutant‐targeting hydrogel scavenger is reported for water remediation with both high selectivity and multiple adsorbability for several pollutants, and with strong affinity and good recyclability through rationally integrating the advantages of multiple functional materials. In the scavenger, aptamers fold into binding pockets to accommodate the molecular structure of pollutants to afford perfect selectivity, and Janus nanoparticles with antibacterial function as well as anisotropic surfaces to immobilize multiple aptamers allow for simultaneously handling different kinds of pollutants. The scavenger exhibits high efficiencies in removing pollutants from water and it can be easily recycled for many times without significant loss of loading capacities. Moreover, the residual concentrations of each contaminant are well below the drinking water standards. Thermodynamic behavior of the adsorption process is investigated and the rate‐controlling process is determined. Furthermore, a point of use device is constructed and it displays high efficiency in removing pollutants from environmental water. The scavenger exhibits great promise to be applied in the next generation of water purification systems.