Isolation Of Aptamers Research Articles

Isolation and characterization of DNA aptamers against the HlyE antigen of Salmonella Typhi

Aptamers have emerged as prominent ligands in clinical diagnostics because they provide various advantages over antibodies, such as quicker generation time, reduced manufacturing costs, minimal batch-to-batch variability, greater modifiability, and improved thermal stability. In the present study, we isolated and characterized DNA aptamers that can specifically bind to the hemolysin E (HlyE) antigen of Salmonella Typhi for future development of typhoid diagnostic tests. The DNA aptamers against Salmonella Typhi HlyE were isolated using systematic evolution of ligands by exponential enrichment (SELEX), and their binding affinity and specificity were assessed utilizing enzyme-linked oligonucleotide assay (ELONA). A total of 11 distinct aptamers were identified, and the binding affinities and species selectivities of the three most probable aptamers were determined. Kd values were obtained in the nanomolar range, with the highest affinity of 83.6 nM determined for AptHlyE97. In addition, AptHlyE11, AptHlyE45 and AptHlyE97 clearly distinguished S. Typhi HlyE from other tested bacteria, such as Salmonella Paratyphi A, Salmonella Paratyphi B, Shigella flexneri, Klebsiella pneumonia and Escherichia coli, therefore displaying desirable specificity. These novel aptamers could be used as diagnostic ligands for the future development of inexpensive and effective point-of-care tests for typhoid surveillance, especially in developing countries of the tropics and subtropics.

Isolation and characterization of ssDNA aptamers against BipD antigen of Burkholderia pseudomallei

BackgroundMelioidosis is difficult to diagnose due to its wide range of clinical symptoms. The culture method is time-consuming and less sensitive, emphasizing the importance of rapid and accurate diagnostic tests for melioidosis. Burkholderia invasion protein D (BipD) of Burkholderia pseudomallei is a potential diagnostic biomarker. This study aimed to isolate and characterize single-stranded DNA aptamers that specifically target BipD. MethodsThe recombinant BipD protein was produced, followed by isolation of BipD-specific aptamers using Systematic Evolution of Ligands by EXponential enrichment. The binding affinity and specificity of the selected aptamers were evaluated using Enzyme-Linked Oligonucleotide Assay. ResultsThe fifth SELEX cycle showed a notable enrichment of recombinant BipD protein-specific aptamers. Sequencing analysis identified two clusters with a total of seventeen distinct aptamers. AptBipD1, AptBipD13, and AptBipD50 were chosen based on their frequency. Among them, AptBipD1 exhibited the highest binding affinity with a Kd value of 1.0 μM for the recombinant BipD protein. Furthermore, AptBipD1 showed significant specificity for B. pseudomallei compared to other tested bacteria. ConclusionAptBipD1 is a promising candidate for further development of reliable, affordable, and efficient point-of-care diagnostic tests for melioidosis.

Rapid Nuclease-Assisted Selection of High-Affinity Small-Molecule Aptamers.

Aptamers are nucleic acid bioreceptors that have been widely utilized for a variety of biosensing applications, including in vivo detection methods that would not be possible with antibody-based systems. However, it remains challenging to generate high-quality aptamers for small molecule targets, particularly for use under physiological conditions. We present a highly effective aptamer selection technology for small-molecule targets that utilizes the nuclease EcoRI to remove nonspecific or weakly binding sequences in solution phase, rapidly enriching high-affinity target binders within just a few rounds of selection. As proof-of-concept, we used our nuclease-assisted SELEX (NA-SELEX) method to isolate aptamers for a synthetic cannabinoid, AB-FUBINACA. Within five rounds, we identified two highly specific aptamers that exhibit nanomolar affinity at physiological temperature. We also demonstrate the robustness and reproducibility of NA-SELEX by performing the same selection experiment with fresh reagents and libraries, obtaining the same two aptamers as well as two other high-quality aptamer candidates. Finally, we compare NA-SELEX against a conventional library-immobilized SELEX screen for AB-FUBINACA using the same screening conditions, identifying aptamers with 25-100-fold weaker affinity after 11 rounds of selection. NA-SELEX therefore could be an effective selection method for the isolation of high-quality aptamers for small-molecule targets.

Peptide-based CE-SELEX enables convenient isolation of aptamers specifically recognizing CD20-expressing cells

The CD20 antigen is a key target for several diseases including lymphoma and autoimmune diseases. For over 20 years, several monoclonal antibodies were developed to treat CD20-related disorders. As many therapeutic proteins, their clinical use is however limited due to their nature with a costly biotechnological procedure and side effects such as the production of anti-drug neutralizing antibodies. Nucleic acid aptamers have some advantages over mAbs and are currently investigated for clinical use. We herein report the selection of DNA aptamer by using a peptide-based CE-SELEX (Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment) method. It was demonstrated that these aptamers bind specifically a CD20-expressing human cell line, with Kd estimated from isothermal titration calorimetry experiments in the micromolar range. This study demonstrates that the CE-SELEX is suitable as alternative method to the conventional Cell-SELEX to discover new cell-targeting compounds.

Isolation of aptamers with excellent cross-reactivity and specificity to sulfonamides towards a ratiometric fluorescent aptasensor for the detection of nine sulfonamides in seafood

Sulfonamides (SAs) is a class of antibiotics that extensively used for treating infectious diseases in livestock industries and aquaculture. Thus, it is urgent need to obtain the bio-receptor, which has excellent cross-reactivity and specificity to SAs, for developing high-throughput methods for the determination of multiple SAs even all commonly-used SAs, to realize the quick screening/detection of total SAs in animal-derived foods. We herein isolated several SAs-specific cross-reactive aptamers by using a library-immobilized SELEX with multi-SAs parallel selection strategy. Two of the isolated aptamers (Sul-01 and Sul-04) can specifically recognize and bind seven SAs respectively with higher binding affinity and no interference of non-sulfonamide antibiotics, and thus can be applied as bio-receptors for developing high-throughput aptasensors for the quick screening/detection of multiple SAs. By using the mixture of Sul-01 and Sul-04 as bio-receptor, a ratiometric fluorescent aptasensor was created for the quick detection of nine SAs including sulfamethoxydiazine (SMD), sulfapyridine (SPD), sulfaquinoxaline (SQ), sulfathiazole (ST), sulfamonomethoxine (SMM), sulfamerazine (SMR), sulfaguanidine (SG), sulfamethazine (SMZ) and sulfadiazine (SD) with a detection limit (LOD) of 0.10–0.50 μM, or total of above nine SAs with a LOD of 0.20 μM. The fluorescent aptasensor was successfully applied to detect each or total of SMD, SPD, SQ, ST, SMM, SMR, SG, SMZ and SD in fish samples with a recovery of 83 %–92 % and a relative standard deviation (RSD, n = 5) < 5 %. This study not only provided several promising bio-receptors for the development of diverse high-throughput aptasensors to achieve the quick screening of multiple SAs residues, but also provided a simple, stable and sensitive method for the quick screening of SMD, SPD, SQ, ST, SMM, SMR, SG, SMZ and SD in seafood.

Rapid and Sensitive Detection of Inactivated SARS-CoV-2 Virus via Fiber-Optic and Electrochemical Impedance Spectroscopy Based Aptasensors.

The development of rapid detection tools for viruses is vital for the prevention of pandemics and biothreats. Aptamers that target inactivated viruses are attractive for sensors due to their improved biosafety. Here, we evaluated a DNA aptamer (named as 6.9) that specifically binds to the inactivated SARS-CoV-2 virus with a low dissociation constant (KD = 9.6 nM) for the first time. Based on aptamer 6.9, we developed a fiber-optic evanescent wave (FOEW) biosensor. Inactivated SARS-CoV-2 and the Cy5.5-tagged short complementary strand competitively bound with the aptamer immobilized on the surface of the sensor. The detection of the inactivated SARS-CoV-2 virus was realized within six minutes with a limit of detection (LOD, S/N = 3) of 740 fg/mL. We also developed an electrochemical impedance aptasensor which exhibited an LOD of 5.1 fg/mL and high specificity. We further demonstrated that the LODs of the FOEW and electrochemical impedance aptasensors were, respectively, more than 1000 and 100,000 times lower than those of commercial colloidal gold test strips. We foresee that the facile aptamer isolation process and sensor design can be easily extended for the detection of other inactivated viruses.

Cost-effective evaluation of Aptamer candidates in SELEX-based Aptamer isolation

Aptamers are short, single-stranded nucleic acids with high affinity and specificity for various targets, making them valuable in diagnostics and therapeutics. Their isolation traditionally involves a time-consuming and costly process called SELEX. While SELEX methods have evolved to improve binding and amplification, the crucial step of aptamer identification from sequencing data remains expensive and often overlooked. Common identification methods require modification of aptamer candidates with labels like biotin or fluorescent dyes, which becomes costly and cumbersome for high-throughput sequencing data. This paper presents an efficient and cost-effective approach to streamline aptamer identification. It employs asymmetric polymerase chain reaction (PCR) to generate modified single-stranded DNA copies of aptamer candidates, simplifying the modification process. By using excess modified forward primers and limited reverse primers, this method reduces costs since only unmodified candidates need to be synthesized initially. The approach was demonstrated with an IgE protein aptamer and successfully applied to identify aptamers from a pool of 12 candidates against a monoclonal antibody. The validity of the results was further confirmed through the direct synthesis of fluorophore-conjugated aptamer candidates, yielding consistent outcomes while reducing the cost by threefold. This approach addresses a critical bottleneck in aptamer discovery by significantly reducing the time and cost associated with aptamer identification, facilitating aptamer-based research and making aptamers more accessible for various applications in diagnostics and therapeutics.

Rapid isolation of anti-idiotype aptamers for quantification of human monoclonal antibodies against SARS-CoV-2 spike protein

Therapeutic antibodies that block viral entry have already proven to be important, first line drugs for treatments of viral infections. In the case of SARS-CoV-2, combinations of multiple therapeutic antibodies may need to be rapidly identified and formulated in a way that blocks each new, predominant variant of the virus. For efficient introduction of any new antibody combination into patients, it is important to be able to monitor patient-specific pharmacokinetics of individual antibodies, which would include the time course of their specific capacity to block the viral spike proteins. Here, we present three examples of microfluidic-based rapid isolation of companion reagents useful for establishing combination antibody therapies. These reagents are specific three-dimensional imprints of variable regions of individual human monoclonal antibodies against the -spike protein of SARS-CoV-2 virus in the form of oligonucleotide-based ligands (aptamers). We implement these anti-idiotypic aptamers as bioreceptors in graphene-based field-effect transistor sensors to accomplish label free, rapid, and sensitive detection of matching antibodies within minutes. Through this work we have demonstrated the general applicability of anti-idiotype aptamers as capture reagents in quantification of active forms of monoclonal antibodies in complex biological mixtures.

Aptamers selected and identified as a colorimetric dual-mode sensing platform for selective and sensitive detection of capsaicin

Capsaicin (CAP), a prominent indicator, has been identified for detecting the gutter oil in adulterated edible oils. In view of the limited availability of efficient and reliable methods for detecting gutter oil, the present study employed the Capture-SELEX technology, which utilizes a high-throughput sequencing (HTS) platform, to facilitate the precise tracking of screening rounds and facilitate the isolation of aptamers with specific affinity for CAP. Isothermal titration calorimetry (ITC) determined the highest affinity and Kd value of 15.5 ± 4.69 nM for aptamer Cap-1. A colorimetric dual-mode aptasensor was designed for CAP detection based on graphene oxide (GO)@gold nanoparticles (AuNPs) composite. A high-sensitivity assay can be achieved using a Synergy™ H1 multimode zymography instrument and a smartphone. Under optimal conditions, the colorimetric dual-mode aptasensor has a detection range of 0.5 ∼ 120 ng mL−1 and a low limit of detection (LOD) of 0.14 ng mL−1. The quantitative recovery of the sensor for CAP in edible oil was 86.25∼98.12 %. Compared with the conventional single-mode aptasensor, the dual-mode aptasensor has independent signal conversion, which provides suitable detection solutions for different scenarios and improves detection accuracy and reliability.

A functional group-guided approach to aptamers for small molecules.

Aptameric receptors are important biosensor components, yet our ability to identify them depends on the target structures. We analyzed the contributions of individual functional groups on small molecules to binding within 27 target-aptamer pairs, identifying potential hindrances to receptor isolation-for example, negative cooperativity between sterically hindered functional groups. To increase the probability of aptamer isolation for important targets, such as leucine and voriconazole, for which multiple previous selection attempts failed, we designed tailored strategies focused on overcoming individual structural barriers to successful selections. This approach enables us to move beyond standardized protocols into functional group-guided searches, relying on sequences common to receptors for targets and their analogs to serve as anchors in regions of vast oligonucleotide spaces wherein useful reagents are likely to be found.

Recent Progress in Functional-Nucleic-Acid-Based Fluorescent Fiber-Optic Evanescent Wave Biosensors.

Biosensors capable of onsite and continuous detection of environmental and food pollutants and biomarkers are highly desired, but only a few sensing platforms meet the "2-SAR" requirements (sensitivity, specificity, affordability, automation, rapidity, and reusability). A fiber optic evanescent wave (FOEW) sensor is an attractive type of portable device that has the advantages of high sensitivity, low cost, good reusability, and long-term stability. By utilizing functional nucleic acids (FNAs) such as aptamers, DNAzymes, and rational designed nucleic acid probes as specific recognition ligands, the FOEW sensor has been demonstrated to be a general sensing platform for the onsite and continuous detection of various targets ranging from small molecules and heavy metal ions to proteins, nucleic acids, and pathogens. In this review, we cover the progress of the fluorescent FNA-based FOEW biosensor since its first report in 1995. We focus on the chemical modification of the optical fiber and the sensing mechanisms for the five above-mentioned types of targets. The challenges and prospects on the isolation of high-quality aptamers, reagent-free detection, long-term stability under application conditions, and high throughput are also included in this review to highlight the future trends for the development of FOEW biosensors capable of onsite and continuous detection.

In-silico selection employing rigiddocking and moleculardynamic simulation in selecting DNA aptamers against androgen receptor.

Aptamers are a class of single-stranded (ss) nucleic acid molecules generated through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) that involves iterations of time-consuming and tedious selection, amplification, and enrichment steps. To compensate for the drawbacks of conventional SELEX, we have devised an in-silico methodology that facilitates a cost-effective and facile manner of aptamer selection. Here, we report the isolation of DNA aptamers against androgen receptors (ARs) using androgen response elements (ARE) that possess natural affinity toward AR. A virtual library of ARE sequences was prepared and subjected to a stringent selection criterion to generate a sequence pool having stable hairpin conformationsandhighGCcontent. The 3D-structures of the selected ss AREs were modeled and screened through rigid docking and molecular dynamic (MD) simulation to examine their potency as potential AR binders. The predicted sequences were further validated using direct enzyme-linked aptasorbent assay (ELASA),whichincludesthemeasurementoftheir binding affinity, specificity, and target discrimination properties under complex biological enviroments. A short, 15 nucleotides (nts), ssDNA aptamer,termedARapt1withtheestimatedKdvalueof5.5±3nm, was chosen as the mostprominent aptamer against AR based on the coherence of both the in-silico and in-vitro evaluation results. The high target-binding affinity and selectivity of ARapt1 signify its potential use as a versatile tool in diagnostic applications relevant to prostate cancer and related diseases.

Development of multi-reactive aptamers for Cronobacter spp. using the sequential partitioning method to detect them in powdered infant formula

Cronobacter spp. are opportunistic foodborne pathogens typically detected in contaminated powdered infant formula (PIF). Thus, the rapid detection and control of Cronobacter spp. are required to prevent outbreaks, necessitating the development of specific aptamers. In this study, we isolated aptamers specific to all seven species of Cronobacter (C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. dublinensis, C. condimenti, and C. universalis) using a newly proposed sequential partitioning method. This method avoids the repeated enrichment steps, reducing the total aptamer selection time compared with the conventional systematic evolution of ligands by the exponential enrichment (SELEX) process. We isolated four aptamers showing high affinity and specificity for all seven species of Cronobacter, with dissociation constants of 3.7–86.6 nM. This represents the first successful isolation of aptamers for multiple targets using the sequential partitioning method. Further, the selected aptamers could effectively detect Cronobacter spp. in contaminated PIF.

Applications and future of aptamers that achieve rapid-onset anticoagulation.

In this short Perspective, we discuss the history of, and recent progress toward, the development of aptamers that can serve as rapid onset anticoagulants during cardiopulmonary bypass (CPB), extracorporeal membrane oxygenation (ECMO), and catheter-based diagnostic and interventional procedures, several million of which are performed each year worldwide. Aptamer anticoagulants provide potent and antidote-controllable anticoagulation and have low immunogenicity. New methods of aptamer isolation and engineering have not only improved the quality of aptamers, but also accelerated their development. Unfortunately, no aptamer identified to date can produce an anticoagulant effect as potent as that produced by unfractionated heparin (UFH), the standard anticoagulant for CPB. We have suggested several possible strategies to amplify the anticoagulant potency of existing aptamer anticoagulants.

Systematic Study of in Vitro Selection Stringency Reveals How To Enrich High-Affinity Aptamers.

Aptamers are oligonucleotide receptors with great potential for sensing and therapeutic applications. They are isolated from random libraries through an in vitro method termed systematic evolution of ligands by exponential enrichment (SELEX). Although SELEX-based methods have been widely employed over several decades, many aspects of the experimental process remain poorly understood in terms of how to adjust the selection conditions to obtain aptamers with the desired set of binding characteristics. As a result, SELEX is often performed with arbitrary parameters that tend to produce aptamers with insufficient affinity and/or specificity. Having a better understanding of these basic principles could increase the likelihood of obtaining high-quality aptamers. Here, we have systematically investigated how altering the selection stringency in terms of target concentration─which is essentially the root source of selection pressure for aptamer isolation─affects the outcome of SELEX. By performing four separate trials of SELEX for the same small-molecule target, we experimentally prove that the use of excessively high target concentrations promotes enrichment of low-affinity binders while also suppressing the enrichment of high-affinity aptamers. These findings should be broadly applicable across SELEX methods, given that they share the same core operating principle, and will be crucial for guiding selections to obtain high-quality aptamers in the future.

Microfluidic isolation of aptamers with affinity towards multiple myeloma monoclonal immunoglobulins.

Multiple myeloma (MM) is a bone marrow cancer of resident plasma cells that affects 125,000 patients in the U.S. with about 30,000 new cases per year. Its signature is the clonal proliferation of a single plasma cell that secretes a patient specific monoclonal immunoglobulin (M-Ig). Targeting the M-Ig in patient serum could allow sensitive and noninvasive identification of minimal residual disease in multiple myeloma. Aptamers, which are single-stranded oligonucleotides with affinity and specificity to a target molecule, have recently been introduced as affinity reagents for recognition of MM M-Igs. Here we exploit microfluidic SELEX technology to enable rapid and efficient generation of aptamers against M-Ig proteins from MM patients. We first characterize the technology by isolating aptamers with affinity towards the monoclonal antibody rituximab as a model M-Ig and then apply the technology to isolating aptamers specifically targeting M-Igs obtained from serum samples of MM patients. We demonstrate that high-affinity DNA aptamers (KD < 50 nM) for M-Ig proteins from a patient sample could be isolated via microfluidic SELEX within approximately 12h and using less than 100 micrograms of patient M-Ig. Such aptamers can potentially be used in personalized monitoring of minimal residual disease in MM patients.

Surface acoustic wave-assisted microfluidic isolation of aptamers.

Aptamers are synthetic single-stranded nucleic acid molecules that bind to biochemical targets with high affinity and specificity. The method of systematic evolution of ligands by exponential enrichment (SELEX) is widely used to isolate aptamers from randomized oligonucleotides. Recently, microfluidic technology has been applied to improve the efficiency and reduce the cost in SELEX processes. In this work, we present an approach that exploits surface acoustic waves to improve the affinity selection process in microfluidic SELEX. Acoustic streaming is used to enhance the interactions of the solution-based oligonucleotide molecules with microbead-immobilized target molecules, allowing the identification of high-affinity aptamer candidates in a more efficient manner. For demonstration, a DNA aptamer is isolated within three rounds of selection in 5 h to specifically bind to immunoglobulin E, a representative target protein, with an equilibrium dissociation constant of approximately 22.6 nM.

Aptamers Targeting Cardiac Biomarkers as an Analytical Tool for the Diagnostics of Cardiovascular Diseases: A Review

The detection of cardiac biomarkers is used for diagnostics, prognostics, and the risk assessment of cardiovascular diseases. The analysis of cardiac biomarkers is routinely performed with high-sensitivity immunological assays. Aptamers offer an attractive alternative to antibodies for analytical applications but, to date, are not widely practically implemented in diagnostics and medicinal research. This review summarizes the information on the most common cardiac biomarkers and the current state of aptamer research regarding these biomarkers. Aptamers as an analytical tool are well established for troponin I, troponin T, myoglobin, and C-reactive protein. For the rest of the considered cardiac biomarkers, the isolation of novel aptamers or more detailed characterization of the known aptamers are required. More attention should be addressed to the development of dual-aptamer sandwich detection assays and to the studies of aptamer sensing in alternative biological fluids. The universalization of aptamer-based biomarker detection platforms and the integration of aptamer-based sensing to clinical studies are demanded for the practical implementation of aptamers to routine diagnostics. Nevertheless, the wide usage of aptamers for the diagnostics of cardiovascular diseases is promising for the future, with respect to both point-of-care and laboratory testing.

Aptamers as promising nanotheranostic tools in the COVID-19 pandemic era.

The emergence of SARS‐COV‐2, the causative agent of new coronavirus disease (COVID‐19) has become a pandemic threat. Early and precise detection of the virus is vital for effective diagnosis and treatment. Various testing kits and assays, including nucleic acid detection methods, antigen tests, serological tests, and enzyme‐linked immunosorbent assay (ELISA), have been implemented or are being explored to detect the virus and/or characterize cellular and antibody responses to the infection. However, these approaches have inherent drawbacks such as nonspecificity, high cost, are characterized by long turnaround times for test results, and can be labor‐intensive. Also, the circulating SARS‐COV‐2 variant of concerns, reduced antibody sensitivity and/or neutralization, and possible antibody‐dependent enhancement (ADE) have warranted the search for alternative potent therapeutics. Aptamers, which are single‐stranded oligonucleotides, generated artificially by SELEX (Evolution of Ligands by Exponential Enrichment) may offer the capacity to generate high‐affinity neutralizers and/or bioprobes for monitoring relevant SARS‐COV‐2 and COVID‐19 biomarkers. This article reviews and discusses the prospects of implementing aptamers for rapid point‐of‐care detection and treatment of SARS‐COV‐2. We highlight other SARS‐COV‐2 targets (N protein, spike protein stem‐helix), SELEX augmented with competition assays and in silico technologies for rapid discovery and isolation of theranostic aptamers against COVID‐19 and future pandemics. It further provides an overview on site‐specific bioconjugation approaches, customizable molecular scaffolding strategies, and nanotechnology platforms to engineer these aptamers into ultrapotent blockers, multivalent therapeutics, and vaccines to boost both humoral and cellular immunity against the virus.This article is categorized under:Therapeutic Approaches and Drug Discovery > Emerging TechnologiesDiagnostic Tools > BiosensingTherapeutic Approaches and Drug Discovery > Nanomedicine for Infectious DiseaseTherapeutic Approaches and Drug Discovery > Nanomedicine for Respiratory Disease

Directed Evolution of Aptamer Discovery Technologies.

Although antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our laboratory to accelerate the discovery of high-quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multiparametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrixes such as serum. The third method is an alkyne-azide chemistry ("click chemistry")-based particle display (click-PD) that enables the generation and screening of "non-natural" aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Finally, we describe the non-natural aptamer array (N2A2) platform in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in high throughput, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized for affinity and specificity in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.