SELEX Procedure Research Articles

Generation of epitope-specific hCG aptamers through a novel targeted selection approach.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone used as a biomarker for several medical conditions, including pregnancy, trophoblastic and nontrophoblastic cancers. Most commercial hCG tests rely on a combination of antibodies, one of which is usually specific to the C-terminal peptide of the β-subunit. However, cleavage of this region in many hCG degradation variants prevents rapid diagnostic tests from quantifying all hCG variants in serum and urine samples. An epitope contained within the core fragment, β1, represents an under-researched opportunity for developing immunoassays specific to most variants of hCG. In the study described here, we report on a SELEX procedure tailored towards the identification of two pools of aptamers, one specific to the β-subunit of hCG and another to the β1 epitope within it. The described SELEX procedure utilized antibody-blocked targets, which is an underutilized strategy to exert negative selection pressure and in turn direct aptamer enrichment to a specific epitope. We report on the first aptamers, designated as R4_64 and R6_5, each capable of recognising two distinct sites of the hCG molecule-the β-subunit and the (presumably) β1-epitope, respectively. This study therefore presents a new SELEX approach and the generation of novel aptamer sequences that display potential hCG-specific biorecognition.

A highly specific aptamer for the SARS-CoV-2 spike protein from the authentic strain.

DNA aptamers are oligonucleotides that specifically bind to target molecules, similar to how antibodies bind to antigens. We identified an aptamer named MEZ that is highly specific to the receptor-binding domain, RBD, of the SARS-CoV-2 spike protein from the Wuhan-Hu-1 strain. The SELEX procedure was utilized to enrich the initial 31-mer oligonucleotide library with the target aptamer. The aptamer identification was performed using the novel protocol based on nanopore sequencing developed in this study. The MEZ aptamer was chemically synthesized and tested for binding with the SARS-CoV-2 RBD of the spike protein from different strains. The Kd is 6.5 nM for the complex with the RBD from the Wuhan-Hu-1 strain, which is comparable with known aptamers; the advantage is that the MEZ aptamer is smaller than known analogs. The proposed aptamer is highly selective for the RBD protein from the Wuhan-Hu-1 strain and does not form complexes with the RBD from Beta, Delta and Omicron strains. Experimental and theoretical studies together revealed the molecular mechanism of aptamer binding. The aptamer occupies the same binding site as ACE2 when bound to the RBD. The 3'-end of the MEZ aptamer is important for complex formation and is responsible for the discrimination of the RBD protein from a specific strain. The 5'-end is responsible for the formation of a loop in the 3D structure of the aptamer, which is important for proper binding.

Growth and Storage Study of Streptococcus agalactiae as a Potential Target in Whole-Cell Based ‘Systematic Evolution of Ligands by Exponential Enrichment (SELEX)’ Procedure

Streptococcus agalactiae is a pathobiont that causes severe invasive infections, especially in fishponds or freshwater aquatic organisms. Streptococcus outbreak in cultured fishponds causes a negative impact on the production of fishes, especially tilapias. Therefore, it is essential to develop a reliable method for on-site monitoring of S. agalactiae in fishponds. This study aims to investigate a local S. agalactiae strain TP540K as a potential target for DNA aptamer in a whole-cell based ‘systematic evolution of ligands by exponential enrichment (SELEX)’ procedure. Before this procedure, the bacterial growth profile of S. agalactiae strain TP540K was studied. Besides, the viability of S. agalactiae strain TP540K was compared between different storage solutions (glycerol, phosphate buffer saline, distilled water) and temperatures (-20, 4 and 27 °C). In this study, S. agalactiae strain TP540K entered the log phase at 1.5 h of incubation time, thus the cells were collected at 2 h of incubation time with an OD600 value of 0.436 to proceed to SELEX process. Moreover, glycerol stock (20%, -20 °C) was the best storage condition for preservation of S. agalactiae strain TP540K until Day 84 with a cell viability of 2.48 x 107 CFU/mL. With the findings obtained, S. agalactiae strain TP540K could be utilised as a target for future study in DNA aptamer development through whole-cell based SELEX procedure, which could be useful for future investigations to overcome Streptococcus outbreak in the Malaysian aquaculture industry.

Novel Aptamers for the Reactivation of Latent HIV.

A "Shock and Kill" strategy has been proposed to eradicate the HIV latent viral reservoir. Effective Latency Reversal Agents (LRA) are a key requirement for this strategy. The search for LRAs with a novel mechanism of action is ongoing. This is the first study to propose aptamers for the reactivation of HIV. The purpose of this study was to identify an aptamer that potentially reactivates HIV via the NF-κβ pathway, specifically by binding to IkB and releasing NF-κβ. Aptamer selection was performed at Aptus Biotech (www.aptusbiotech.es), using ikB human recombinant protein with His tag bound to Ni-NTA agarose resin using the SELEX procedure. Activation of NF-κβ was measured by SEAP Assay. HIV reactivation was measured in JLat cells using a BD FACS-Canto™ II flow cytometer. All flow cytometry data were analyzed using Kaluza analyzing software. Clones that had equivalent or greater activation than the positive control in the SEAP assay were regarded as potential reactivators of the NF-κβ pathway and were sequenced. The three ikb clones namely R6-1F, R6-2F, and R6-3F were found to potentially activate the NF-κβ pathway. Toxicity was determined by exposing lymphocytes to serial dilutions of the aptamers; the highest concentration of the aptamers that did not decrease viability by > 20% was used for the reactivation experiments. The three novel aptamers R6-1F, R6-2F, and R6-3F resulted in 4,07%, 6,72% and 3,42% HIV reactivation, respectively, while the untreated control showed minimal (<0.18%) fluorescence detection. This study demonstrated the reactivation of latent HIV by aptamers that act via the NF-κβ pathway. Although the effect was modest and unlikely to be of clinical benefit, future studies are warranted to explore ways of enhancing reactivation.

Foot-and-mouth disease virus: DNA aptamer selection for the 3ABC protein

Foot-and-mouth disease (FMD) is a devastating livestock disease caused by foot-and-mouth disease virus (FMDV), a member of the Picornaviridae family. The 3ABC is a non-structural protein of FMDV, produced during viral replication and absent from inactivated FMD vaccines. Nucleic acid aptamers are DNA or RNA oligonucleotides capable of binding with high specificity and affinity to a molecular target. The aim of this study was to obtain DNA aptamers specific for 3ABC protein with a view of their application in the FMD diagnosis.Aptamers are usually obtained through SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. In this study, an aptamer (termed FMDV1) was selected by a variation of this technique called Capillary Electrophoresis SELEX (CE-SELEX). The FMDV1 aptamer showed high binding affinity to the 3ABC protein with Kd value in the nano molar range: 22.69 ± 1.79 nM. The FMDV1 aptamer binding to 3ABC was significantly higher when compared with the BSA protein, used as control, demonstrating its specificity.

Connective tissue growth factor-targeting DNA aptamer suppresses pannus formation as diagnostics and therapeutics for rheumatoid arthritis.

Connective tissue growth factor (CTGF) has been recently acknowledged as an ideal biomarker in the early disease course, participating in the pathogenesis of pannus formation in rheumatoid arthritis (RA). However, existing approaches for the detection of or antagonist targeting CTGF are either lacking or unsatisfactory in the diagnosis and treatment of RA. To address this, we synthesized and screened high-affinity single-stranded DNA aptamers targeting CTGF through a protein-based SELEX procedure. The structurally optimized variant AptW2-1-39-PEG was characterized thoroughly for its high-affinity (KD 7.86 nM), sensitivity (minimum protein binding concentration, 2 ng), specificity (negative binding to other biomarkers of RA), and stability (viability-maintaining duration in human serum, 48 h) properties using various biochemical and biophysical assays. Importantly, we showed the antiproliferative and antiangiogenic activities of the aptamers obtained using functional experiments and further verified the therapeutic effect of the aptamers on joint injury and inflammatory response in collagen-induced arthritis (CIA) mice, thus advancing this study into actual therapeutic application. Furthermore, we revealed that the binding within AptW2-1-39-PEG/CTGF was mediated by the thrombospondin 1 (TSP1) domain of CTGF using robust bioinformatics tools together with immunofluorescence. In conclusion, our results revealed a novel aptamer that holds promise as an additive or alternative approach for CTGF-targeting diagnostics and therapeutics for RA.

Towards the Selection of Single‐Stranded DNA Molecular Recognition Element Against Cyanotoxin L‐BMAA

Global warming, anthropogenic pressure and eutrophication contribute to the formation and persistence of cyanobacterial blooms in aquatic environments. Cyanotoxins produced during these blooms represent a threat to ecosystems, commercial industries and human health. B‐N‐methylamino‐L‐alanine (L‐BMAA) is a neurotoxic amino acid that has been linked to neurodegenerative diseases such as Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC), Alzheimer’s disease (AD) and Amyotrophic Lateral Sclerosis (ALS). Research has shown that L‐BMAA can act as a glutamate receptor antagonist and can incorporate into proteins occasioning protein misfolding that leads to detrimental processes in brain tissues. Currently, the detection of L‐BMAA is costly, time‐consuming and labor‐intensive. Many research groups around the world are working towards the development of bioanalytical methods to improve the detection of these toxins, including the development of molecular biorecognition elements (MREs) that can provide analyte specificity in biosensing applications. Among MREs, aptamers are well‐known to possess an extraordinary target recognition due to the formation of unique three‐dimensional structures capable of binding specifically to their respective targets.We aim to perform an in vitro selection of a single‐stranded DNA (ssDNA) aptamer that can act as an MRE against L‐BMAA, using the systematic evolution of ligands by exponential enrichment (SELEX) technique. We have worked on the optimization of incubation conditions, PCR amplification parameters and the generation of ssDNA using two different ssDNA libraries, with randomized regions of varying lengths. To facilitate the separation of bound and unbound ssDNA after incubation with the target, we modified L‐BMAA with a biotinylated linker to immobilize it in streptavidin‐coated magnetic beads. For each library, we have identified the appropriate annealing temperature, primer concentrations and number of cycles to favor the amplification of the main product while minimizing by‐product formation. The most challenging step of the SELEX procedure has been the generation and purification of ssDNA in sufficient amounts to continue to the next round of selection. To achieve this, we have tried four main methods: exonuclease digestion, size separation derived from uneven primers, asymmetric PCR and alkaline denaturation using sodium hydroxide. Hitherto, we have completed the first round of selection with both libraries, by performing large‐scale PCR reactions and alkaline denaturation followed by ethanol precipitation to generate the ssDNA. We are working in the first round of negative selection to discard those sequences with affinity to the immobilization linker and enrich only those that bind exclusively to L‐BMAA. Given that our target is a small molecule, our goal is to conduct a stringent SELEX process to obtain a highly specific MRE that can be incorporated into an environmental sensing device.

High Mannose-Specific Aptamers for Broad-Spectrum Virus Inhibition and Cancer Targeting

High Mannose-Specific Aptamers for Broad-Spectrum Virus Inhibition and Cancer Targeting

FFF-based high-throughput sequence shortlisting to support the development of aptamer-based analytical strategies

Aptamers are biomimetic receptors that are increasingly exploited for the development of optical and electrochemical aptasensors. They are selected in vitro by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, but although they are promising recognition elements, for their reliable applicability for analytical purposes, one cannot ignore sample components that cause matrix effects. This particularly applies when different SELEX-selected aptamers and related truncated sequences are available for a certain target, and the choice of the aptamer should be driven by the specific downstream application. In this context, the present work aimed at investigating the potentialities of asymmetrical flow field-flow fractionation (AF4) with UV detection for the development of a screening method of a large number of anti-lysozyme aptamers towards lysozyme, including randomized sequences and an interfering agent (serum albumin). The possibility to work in native conditions and selectively monitor the evolution of untagged aptamer signal as a result of aptamer-protein binding makes the devised method effective as a strategy for shortlisting the most promising aptamers both in terms of affinity and in terms of selectivity, to support subsequent development of aptamer-based analytical devices.Graphical abstract

Surface plasmon resonance aptasensor for soluble ICAM-1 protein in blood samples.

Intercellular Adhesion Molecule-1 (ICAM-1) is considered to be a cancer biomarker in the assessment of metastatic potential in patients and an early indicator of atherosclerosis. A labelless biosensor based on the surface plasmon resonance (SPR) signal from the specific affinity interaction of an aptamer and a soluble ICAM-1 protein was developed for blood samples. The developed aptasensor provided real-time information on the concentration of the ICAM-1 protein in blood when integrated to a purification step based on a magnetic pull-down separation. The SPR aptasensor was highly specific with a limit of detection of 1.4/0.2 ng ml-1, which was achieved through aptamer-functionalized silica-coated magnetic nanoparticles.

NON-SELEX-BASED IN-SILICO MODELED APTAMERS AGAINST SARS-COV-2 PROTEINS: A SYSTEMATIC REVIEW

Background: The SARS-CoV-2 transmitted COVID-19 pandemics have constituted a worldwide health challenge. Effective diagnosis and treatment of COVID-19 demands that a more reliable and rapid detection protocol should be adopted to replace the real time Polymerase Chain Reaction (RT-PCR) currently in use. Several researches were focused on the possible application of Aptamers and aptamer-based biosensors for swift point-of-care detection of the Covid-19 virus. Target-specific aptamers are generally developed through selection protocol referred to as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Conventionally, computational tools are used for post SELEX analysis, optimization and validation of SELEXdeveloped aptamers. SELEX procedure is however tedious, prohibitive, and time-ineffective. Objective: The aim of this review is to identify studies that present new aptamers modeled in silico against target proteins in SARS-CoV2, high lightening the particular in silico methods employed Methods: Relevant articles that employed in silico approaches in designing Aptamers against SARS-CoV-2 proteins, retrieved from Scopus and PUBMED databases, screened and analyzed. Results: The study revealed that in silico design of aptamer typical entails construction of Aptamer library, target selection, structural modeling of apatmer, molecular docking, optimization and molecular dynamics (MD) simulation. Conclusions: In silico design of Aptamer is therefore a promising time- and cost-effective replacement to SELEX protocol.

Key criteria for engineering mycotoxin binding aptamers via computational simulations: Aflatoxin B1 as a case study.

Due to the difficulties in monoclonal antibody production specific to mycotoxins, aptameric probes have been considered as suitable alternatives. The low efficiency of the SELEX procedure in screening high affinity aptamers for binding mycotoxins as small molecules can be significantly improved through computational techniques. Previously, we designed five new aptamers to aflatoxin B1 (AFB1) based on a known aptamer sequence (Patent: PCT/CA2010/001 292, Apt1) through a genetic algorithm-based in silico maturation strategy and experimentally measured their affinity to the target toxin. Here, integrated molecular dynamic simulation (MDs) studies with molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) analysis to clarify the binding modes, critical interacting nucleic bases and energy component contributions in the six AFB1-binding aptamers. The aptamer F20, which was selected in the first work, showed the best free binding energy and complex stability compared to other aptamers. The trajectory analysis revealed that AFB1 recognized F20 through the groove binding mode along with precise shape complementarity. The MD simulation results revealed that dynamic water intermediate interactions also play a key role in promoting complex stability. According to the MM-PBSA calculations, van der Waals contacts were identified as dominant energy components in all complexes. Interestingly, a high consistency is observed between the experimentally obtained binding affinities of the six aptamers with their free energy solvation. The computational findings, confirmed via previous experiments, highlighted the binding modes, the dynamic hydration of complex components and the total free interacting energy as the crucial criteria in discovering high functional aptameric probes.

HT-SELEX-based identification of binding pre-miRNA hairpin-motif for small molecules

Selective targeting of biologically relevant RNAs with small molecules is a long-standing challenge due to the lack of clear understanding of the binding RNA motifs for small molecules. The standard SELEX procedure allows the identification of specific RNA binders (aptamers) for the target of interest. However, more effort is needed to identify and characterize the sequence-structure motifs in the aptamers important for binding to the target. Herein, we described a strategy integrating high-throughput (HT) sequencing with conventional SELEX followed by bioinformatic analysis to identify aptamers with high binding affinity and target specificity to unravel the sequence-structure motifs of pre-miRNA, which is essential for binding to the recently developed new water-soluble small-molecule CMBL3aL. To confirm the fidelity of this approach, we investigated the binding of CMBL3aL to the identified motifs by surface plasmon resonance (SPR) spectroscopy and its potential regulatory activity on dicer-mediated cleavage of the obtained aptamers and endogenous pre-miRNAs comprising the identified motif in its hairpin loop. This new approach would significantly accelerate the identification process of binding sequence-structure motifs of pre-miRNA for the compound of interest and would contribute to increase the spectrum of biomedical application.

Isolation of Natural DNA Aptamers for Challenging Small-Molecule Targets, Cannabinoids.

Aptamers are nucleic acid-based affinity reagents that are isolated via an in vitro process known as systematic evolution of ligands by exponential enrichment (SELEX). Despite their great potential for a wide range of analytical applications, there are relatively few high-quality small-molecule binding aptamers, especially for "challenging" targets that have low water solubility and/or limited moieties for aptamer recognition. The use of libraries containing chemically modified bases may improve the outcome of some SELEX experiments, but this approach is costly and yields inconsistent results. Here, we demonstrate that a thoughtfully designed SELEX procedure with natural DNA libraries can isolate aptamers with high affinity and specificity for challenging small molecules, including targets for which such selections have previously failed. We first isolate a DNA aptamer with nanomolar affinity and high specificity for (-)-trans-Δ9-tetrahydrocannabinol (THC), a target previously thought to be unsuitable for SELEX with natural DNA libraries. We subsequently isolate aptamers that exhibit high affinity and cross-reactivity to two other challenging targets, synthetic cannabinoids UR-144 and XLR-11, while maintaining excellent specificity against a wide range of non-target interferents. Our findings demonstrate that natural nucleic acid libraries can yield high-quality aptamers for small-molecule targets, and we outline a robust workflow for isolating other such aptamers in future selection efforts.

Development of a CD63 Aptamer for Efficient Cancer Immunochemistry and Immunoaffinity-Based Exosome Isolation.

CD63, a member of transmembrane-4-superfamily of tetraspanin proteins and a highly N-glycosylated type III lysosomal membrane protein, is known to regulate malignancy of various types of cancers such as melanoma and breast cancer and serves as a potential marker for cancer detection. Recently, its important role as a classic exosome marker was also emphasized. In this work, via using a magnetic bead-based competitive SELEX (systematic evolution of ligands by exponential enrichment) procedure and introducing a 0.5 M NaCl as elution buffer, we identified two DNA aptamers (CD63-1 and CD63-2) with high affinity and specificity to CD63 protein (Kd = 38.71 nM and 78.43, respectively). Furthermore, CD63-1 was found to be efficient in binding CD63 positive cells, including breast cancer MDA-MB-231 cells and CD63-overexpressed HEK293T cells, with a medium binding affinity (Kd ~ 100 nM) as assessed by flow cytometry. When immunostaining assay was performed using clinical breast cancer biopsy, the CD63-1 aptamer demonstrated a comparable diagnostic efficacy for CD63 positive breast cancer with commercial antibodies. After developing a magnetic bead-based exosome immunoaffinity separation system using CD63-1 aptamer, it was found that this bead-based system could effectively isolate exosomes from both MDA-MB-231 and HT29 cell culture medium. Importantly, the introduction of the NaCl elution in this work enabled the isolation of native exosomes via a simple 0.5M NaCl incubation step. Based on these results, we firmly believe that the developed aptamers could be useful towards efficient isolation of native state exosomes from clinical samples and various theranostic applications for CD63-positive cancers.

SELEX-based DNA Aptamer Selection: A Perspective from the Advancement of Separation Techniques.

DNA aptamers, which are short, single-stranded DNA sequences that selectively bind to target substances (proteins, cells, small molecules, metal ions), can be acquired by means of the systematic evolution of ligands by exponential enrichment (SELEX) methodology. In the SELEX procedure, one of the keys for the effective acquisition of high-affinity and functional aptamer sequences is the separation stage to isolate target-bound DNA from unbound DNA in a randomized DNA library. In this review, various remarkable advancements in separation techniques for SELEX-based aptamer selection developed in this decade, are described and discussed, including CE-, microfluidic chip-, solid phase-, and FACS-based SELEX along with other methods.

ESTIMATES FROM EVOLUTIONARY ALGORITHMS THEORY APPLIED&#x0D; TO DIRECTED EVOLUTION

The field of evolutionary computation emerged in the area of computer science due to transfer of ideas from biology and developed independently for several decades, enriched with techniques from probability theory, complexity theory and optimization methods. Our aim is to consider how some recent results form the theory of evolutionary computation may be transferred back into biology. It has been noted that&#x0D; the non-elitist evolutionary algorithms optimizing Royal Road fitness functions may be considered as models of evolutionary search for the synthetic enhancer sequences “from scratch”. This problem asks for a tight cluster of supposedly unknown motifs&#x0D; from the initial random (or partially random) set of DNA sequences using SELEX approaches. We apply the upper bounds on the expected hitting time of a target area of genotypic space in order to upper-bound the expected time to finding a sufficiently fit series of motifs in a SELEX procedure. On the other hand, using the theory of evolutionary computation, we propose an upper bound on the expected proportion of the DNA sequences with sufficiently high fitness at a given round of a SELEX procedure. Both approaches are evaluated in computational experiment, using a Royal Road fitness function as a model of the SELEX procedure for regulatory FIS factor&#x0D; binding site.

Aptamers and Antisense Oligonucleotides for Diagnosis and Treatment of Hematological Diseases.

Aptamers or chemical antibodies are single-stranded DNA or RNA oligonucleotides that bind proteins and small molecules with high affinity and specificity by recognizing tertiary or quaternary structures as antibodies. Aptamers can be easily produced in vitro through a process known as systemic evolution of ligands by exponential enrichment (SELEX) or a cell-based SELEX procedure. Aptamers and modified aptamers, such as slow, off-rate, modified aptamers (SOMAmers), can bind to target molecules with less polar and more hydrophobic interactions showing slower dissociation rates, higher stability, and resistance to nuclease degradation. Aptamers and SOMAmers are largely employed for multiplex high-throughput proteomics analysis with high reproducibility and reliability, for tumor cell detection by flow cytometry or microscopy for research and clinical purposes. In addition, aptamers are increasingly used for novel drug delivery systems specifically targeting tumor cells, and as new anticancer molecules. In this review, we summarize current preclinical and clinical applications of aptamers in malignant and non-malignant hematological diseases.

Molecular Epitope Determination of Aptamer Complexes of the Multidomain Protein C-Met by Proteolytic Affinity-Mass Spectrometry.

C-Met protein is a glycosylated receptor tyrosine kinase of the hepatocyte growth factor (HGF), composed of an α and a β chain. Upon ligand binding, C-Met transmits intracellular signals by a unique multi-substrate docking site. C-Met can be aberrantly activated leading to tumorigenesis and other diseases, and has been recognized as a biomarker in cancer diagnosis. C-Met aptamers have been recently considered a useful tool for detection of cancer biomarkers. Herein we report a molecular interaction study of human C-Met expressed in kidney cells with two DNA aptamers of 60 and 64 bases (CLN0003 and CLN0004), obtained using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure. Epitope peptides of aptamer-C-Met complexes were identified by proteolytic affinity-mass spectrometry in combination with SPR biosensor analysis (PROTEX-SPR-MS), using high-pressure proteolysis for efficient digestion. High affinities (KD , 80-510 nM) were determined for aptamer-C-Met complexes, with two-step binding suggested by kinetic analysis. A linear epitope, C-Met (381-393) was identified for CLN0004, while the CLN0003 aptamer revealed an assembled epitope comprised of two peptide sequences, C-Met (524-543) and C-Met (557-568). Structure modeling of C-Met-aptamers were consistent with the identified epitopes. Specificities and affinities were ascertained by SPR analysis of the synthetic epitope peptides. The high affinities of aptamers to C-Met, and the specific epitopes revealed render them of high interest for cellular diagnostic studies.

Perspective on the Future Role of Aptamers in Analytical Chemistry.

It has been almost 30 years since the invention of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology and the description of the first aptamers. In retrospect over the past 30 years, advances in aptamer development and application have demonstrated that aptamers are potentially useful reagents that can be employed in diverse areas within analytical chemistry, biotechnology, biomedicine, and molecular biology. While often touted as artificial antibodies with an ability to be selected for any target, aptamer development, unfortunately, lags behind development of analytical methodologies that employ aptamers, hindering deeper integration into the application of analytical tool development. This perspective covers recent advances in SELEX methodology for improving efficiency of the SELEX procedure and enhancing affinity and specificity of the selected aptamers, what we view as a critical barrier in the future role of aptamers in analytical chemistry. We discuss postselection modifications that can be used for enhancing performance of the selected aptamers in an analytical device by including understanding intermolecular interaction forces in the binding domain. While highlighting promising properties of aptamers that enable several analytical advances, we provide discussion on the challenges of penetration of aptamers in the analytical field.