SELEX Strategy Research Articles

Functional SELEX and Biomedical Applications of Aptamers: Beyond Molecular Recognition

Aptamers, developed through SELEX (systematic evolution of ligands by exponential enrichment), are generally short oligonucleotide molecules with remarkable specificity and binding affinity to diverse biological targets. These molecules have shown promise across such fields as biosensing, molecular diagnostics, and bioimaging. However, while conventional aptamer selection strategies predominantly emphasize binding affinity, they overlook the broader spectrum of potential biological functionalities. This oversight results in aptamers that frequently exhibit limited capacity for direct biological regulation. This inherent limitation in the selection process significantly constrains both the widespread application of aptamers across diverse fields and their therapeutic potential as direct drug candidates. Functional SELEX represents an advancement by refining selection library construction and selection processes to create F‐aptamers that integrate precise molecular recognition with specific biological activities. F‐aptamers hold transformative potential as therapeutic agents, diagnostic tools, and molecular regulators, marking a significant step forward in biomedical applications. This minireview critically assesses recent developments in F‐aptamer SELEX strategies, addressing challenges and exploring opportunities for future research in this dynamic field.

Functional SELEX and Biomedical Applications of Aptamers: Beyond Molecular Recognition

Aptamers, developed through SELEX (systematic evolution of ligands by exponential enrichment), are generally short oligonucleotide molecules with remarkable specificity and binding affinity to diverse biological targets. These molecules have shown promise across such fields as biosensing, molecular diagnostics, and bioimaging. However, while conventional aptamer selection strategies predominantly emphasize binding affinity, they overlook the broader spectrum of potential biological functionalities. This oversight results in aptamers that frequently exhibit limited capacity for direct biological regulation. This inherent limitation in the selection process significantly constrains both the widespread application of aptamers across diverse fields and their therapeutic potential as direct drug candidates. Functional SELEX represents an advancement by refining selection library construction and selection processes to create F‐aptamers that integrate precise molecular recognition with specific biological activities. F‐aptamers hold transformative potential as therapeutic agents, diagnostic tools, and molecular regulators, marking a significant step forward in biomedical applications. This minireview critically assesses recent developments in F‐aptamer SELEX strategies, addressing challenges and exploring opportunities for future research in this dynamic field.

Label-free SELEX of aptamers for ultra-sensitive electrochemical aptasensor detection of amanitin in wild mushrooms

Label-free SELEX of aptamers for ultra-sensitive electrochemical aptasensor detection of amanitin in wild mushrooms

Induction of binding sites for RecA aptamers by differentiated-competition capillary Electrophoresis-SELEX

Induction of binding sites for RecA aptamers by differentiated-competition capillary Electrophoresis-SELEX

Aptamers for mycotoxin recognition in food: Recent advances and future considerations

Aptamers for mycotoxin recognition in food: Recent advances and future considerations

De Novo Evolution of an Antibody-Mimicking Multivalent Aptamer via a DNA Framework.

Multivalent interactions can often endow ligands with more efficient binding performance toward target molecules. Generally speaking, a multivalent aptamer can be constructed via post-assembly based on chemical structural information of target molecules and pre-identified monovalent aptamers derived from traditional systematic evolution of ligands by exponential enrichment (SELEX) technology. However, many target molecules may not have known matched aptamer partners, thus a de novo evolution will be highly desired as an alternative strategy for directed selection of a high-avidity, multivalent aptamer. Here, inspired by the superiority of multivalent interactions between antibodies and antigens, a direct SELEX strategy with a preorganized DNA framework library for an "Antibody-mimicking multivalent aptamer" (Amap) selection to epithelial cell adhesion molecule (EpCAM), a model target protein is reported. The Amap presents a relatively good binding affinity through both aptamer moieties concurrently binding to EpCAM, which has been confirmed by affinity analysis and molecular modeling. Furthermore, dynamic interactions between Amap and EpCAM are directly visualized by magnetic tweezers at the single-molecule level. A nice binding affinity of Amap to EpCAM-positive cancer cells has also been verified, which hints that their Amap-SELEX strategy has the potential to be a new route for de novo evolution of multivalent aptamers.

Generation of an RNA aptamer against LipL32 of Leptospira isolated by Tripartite-hybrid SELEX coupled with in-house Python-aided unbiased data sorting

Generation of an RNA aptamer against LipL32 of Leptospira isolated by Tripartite-hybrid SELEX coupled with in-house Python-aided unbiased data sorting

Evolution of Cell-Type-Specific RNA Aptamers via Live Cell-Based SELEX.

Live cell-based SELEX (Systematic Evolution of Ligand EXponential enrichment) is a promising approach for identifying aptamers that can selectively bind to a cell-surface receptor or recognize a particular target cell population. In particular, it offers a facile selection strategy for some special cell-surface proteins that are originally glycosylated or heavily posttranslationally modified and are unavailable in their native/active conformation after in vitro expression and purification. In this chapter, we describe a generalized procedure for evolution of cell type-specific RNA aptamers targeting a cell membrane bound target by combining the live cell-based SELEX strategy with high-throughput sequencing (HTS) and bioinformatics analysis.

In vitro selection and characterization of ssDNA aptamers by cross-over SELEX and its application for detection of S. Typhimurium

In vitro selection and characterization of ssDNA aptamers by cross-over SELEX and its application for detection of S. Typhimurium

Selection and development of aptamers for pyoverdine

The rhizosphere is a small region surrounding plant roots that is enriched in biochemicals from root exudates and populated with fungi, nematode, and bacteria. Interaction of rhizosphere organisms with plants is mainly promoted by exudates from the roots. Root exudates contain biochemicals that come from primary and secondary metabolisms of plants. These biochemicals attract microbes, which influence plant nutrition. The rhizosphere bacteria (microbiome) are vital to plant nutrient uptake and influence biotic and abiotic stress and pathogenesis. Pseudomonas is a genus of gammaproteobacteria known for its ubiquitous presence in natural habitats and its striking ecological, metabolic, and biochemical diversity. Within the genus, members of the Pseudomonas fluorescens group are common inhabitants of soil and plant surfaces, and certain strains function in the biological control of plant disease, protecting plants from infection by soilborne and aerial plant pathogens. The soil bacterium Pseudomonas protegens Pf‐5 (also known as Pseudomonas fluorescensPf‐5) is a well‐characterized biological strain, which is distinguished by its prolific production of the secondary metabolite, pyoverdine. Knowledge of the distribution of P. fluorescens secretory activity around plant roots is very important for understanding the interaction between P. fluorescens and plants and can be achieved by real time tracking of pyoverdine. To achieve the capability of real‐time tracking in soil, we have used a structure‐switching SELEX strategy to select high affinity ssDNA and 2’ F‐Y‐RNA aptamers with specificity for pyoverdine over other siderophores. Pyoverdines from various strains possess a conserved chromophore along with strain‐specific peptide chain. Some of the isolated aptamers bind to chromophore of pyoverdine. These will identify pyoverdines from a variety of pseudomonads. Identification of aptamers that selectively bind the peptide portion of pyoverdine Pf‐5 is underway. The identified aptamers that are broadly and narrowly specific for pyoverdines or Pf‐5 will be optimized for integration into an electrochemical biosensor to track, in real time, P fluorescens activity and other pseudomonads in and around the rhizosphere.

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

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

Development and analysis of a novel AF11–2 aptamer capable of enhancing the fluorescence of aflatoxin B1

Development and analysis of a novel AF11–2 aptamer capable of enhancing the fluorescence of aflatoxin B1

Real milk sample assisted selection of specific aptamer towards sarafloxacin and its application in establishment of an effective aptasensor

Real milk sample assisted selection of specific aptamer towards sarafloxacin and its application in establishment of an effective aptasensor

Identification of a novel RNA aptamer that selectively targets breast cancer exosomes

Identification of a novel RNA aptamer that selectively targets breast cancer exosomes

Targeting breast cancer exosomes with nucleic aptamers: innovative tools for early diagnosis and therapy

Exosomes are emerging as promising target for early diagnosis and therapy in diff erent oncological conditions including breast cancer (BC). However, the development of tools able to easily and specifi cally target cancer cell-derived exosomes still represent a fundamental issue that is required to realize their clinical utility. Nucleic-acid aptamers are a promising class of structured single stranded oligonucleotides that serve as high affi nity ligands of disease-associated proteins. Given their high potential in diagnosis and therapy, we addressed the development of aptamers specifi c for BC-derived exosomes. To this end, we developed a novel SELEX strategy by using exosomes purifi ed from primary BC cells as positive selection target. By such a strategy we isolated nuclease resistant RNA aptamers able to specifi cally discriminate BC-derived exosomes from those produced by normal cells. Th e best sequences were optimized identifying short molecules (about 30-35 mer) that was characterized as tools for exosome detection. Further, we demonstrated that the developed aptamers inhibited exosome cellular uptake antagonizing cancer exosome-induced cell migration. By proteomic approach we identifi ed possible targets that we are characterizing. Our results underline the great potential of isolated aptamers as tools for the development of innovative strategies for BC early diagnosis and therapy.

The isolation of a DNA aptamer to develop a fluorescent aptasensor for the thiamethoxam pesticide.

Aptamers, which are called chemical antibodies for their high affinity and specificity to targets, have great potential as analytical tools to detect pesticides. In this work, a DNA aptamer for thiamethoxam was isolated by an improved SELEX (systematic evolution of ligands by exponential enrichment) strategy, in which the ssDNA library was fixed on streptavidin-agarose beads through a short biotin labeled complementary strand. After 13 rounds of selection, the random ssDNA pool was successfully enriched. Three sequences were chosen as aptamer candidates through sequencing and analysis and were transformed into fluorescent probes to evaluate their interactions with thiamethoxam. A fluorescent turn-on aptasensor for thiamethoxam based on the best aptamer (FAM-Thi13) and a short quenching strand were further designed and showed a quantitative linear range from 10 to 1000 nM with a detection limit of 1.23 nM for thiamethoxam. Molecular docking and molecular dynamics were used to investigate the binding site of the main probe of the aptasensor (FAM-Thi13) and thiamethoxam. Satisfactory results were also obtained in quantifying thiamethoxam in environmental water samples by the developed fluorescent aptasensor.

Generation of HBsAg DNA aptamer using modified cell-based SELEX strategy.

Aptamers as potential alternatives for antibodies could be employed against hepatitis B surface antigen (HBsAg), the great hallmark and first serological marker in HBV, for further theragnostic applications. Therefore, isolation HBsAg specific aptamer was performed in this study with a modified Cell-SELEX method. HEK293T overexpressing HBsAg and HEK293T as target and control cells respectively, were incubated with single-stranded rounds of DNA library during six SELEX and Counter SELEX rounds. Here, we introduced the new modified Cell-SELEX using deoxyribonuclease I digestion to separate single stranded DNA aptamers against the HBsAg. Characterization and evaluation of selected sequences were performed using flow cytometry analysis. The results led to isolation of 15 different ssDNA clones in six rounds of selection which were categorized to four clusters based on common structural motifs. The evaluation of SELEX progress showed growth in aptamer affinity with increasing in the cycle number. Taken together, the application of modified cell-SELEX demonstrated the isolation of HBsAg-specific ssDNA aptamers with proper affinity. Modified cell-SELEX as an efficient method can shorten the selection procedure and increase the success rate while the benefits of cell-based SELEX will be retained. Selected aptamers could be applied in purification columns, diagnostic kits, and drug delivery system against HBV-related liver cancer.

GOLD SELEX: a novel SELEX approach for the development of high-affinity aptamers against small molecules without residual activity.

GOLD SELEX, a novel SELEX approach has been developed that obviates the need for target immobilization for aptamer development. The approach purely relies on the affinity of the aptamers towards its target, to get detached from the gold nanoparticle (GNP) surface (weak attraction) after binding with its target. Thus, only the completely detached aptamers are selected for the next round of SELEX. This, in-process, also addresses the issue of residual binding and thus improves the sensitivity of the developed aptamers. As a proof of concept for establishing the utility of the approach for small molecules, we have developed aptamers against dichlorvos (DV), a pesticide in just 8 rounds. Using these aptamer candidates, we have developed an aptamer-NanoZyme (GNP having peroxidase mimic activity) based colorimetric assay. The developed aptamer displayed high affinity (Kd in sub micromolar range) and selectivity for DV. The developed assay could detect as low as 15μM DV. The best-performing aptamer was also able to work in real samples like river water and commercial apple juice. The GOLD SELEX approach developed in this study, we believe, can act as a template for future SELEX strategy development and can replace the conventional SELEX strategy.

CRISPR-Mediated Isogenic Cell-SELEX Approach for Generating Highly Specific Aptamers Against Native Membrane Proteins.

The generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most in vitro selection paradigms utilize different cell types for positive and negative rounds of selection (where the positive selection is against a cell that expresses the desired membrane protein and the negative selection is against a cell that lacks the protein). However, this strategy can yield affinity reagents that bind unintended membrane proteins on the target cells. To address this issue, we developed a systematic evolution of ligands by exponential enrichment (SELEX) scheme that utilizes isogenic pairs of cells generated via CRISPR techniques. Using a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the SLC2A1 gene (encoding the GLUT1 glucose transporter) via lipofection with synthetic gRNAs. Cell-SELEX rounds were carried out against wild-type and GLUT1-null cells using a single-strand DNA (ssDNA) library. Next-generation sequencing (NGS) was used to quantify enrichment of prospective binders to the wild-type cells. 10 rounds of cell-SELEX were conducted via simultaneous exposure of ssDNA pools to wild-type and GLUT1-null Caco-2 cells under continuous perfusion. The top binders identified from NGS were validated by flow cytometry and immunostaining for their specificity to the GLUT1 receptor. Our data indicate that highly specific aptamers can be isolated with a SELEX strategy that utilizes isogenic cell lines. This approach may be broadly useful for generating affinity reagents that selectively bind to membrane proteins in their native conformations on the cell surface.

One-Pot SELEX: Identification of Specific Aptamers against Diverse Steroid Targets in One Selection.

Aptamers are well-established biorecognition molecules used in a wide variety of applications for the detection of their respective targets. However, individual SELEX processes typically performed for the identification of aptamers for each target can be quite time-consuming, labor-intensive, and costly. An alternative strategy is proposed herein for the simultaneous identification of different aptamers binding distinct but structurally similar targets in one single selection. This one-pot SELEX approach, using the steroids estradiol, progesterone, and testosterone as model targets, was achieved by combining the benefits of counter-SELEX with the power of next-generation sequencing and bioinformatics analysis. The pools from the last stage of the selection were compared in order to discover sequences with preferential abundance in only one of the pools. This led to the identification of aptamer candidates with potential specificity to a single steroid target. Binding studies demonstrated the high affinity of each selected aptamer for its respective target, and low nanomolar range dissociation constants calculated were similar to those previously reported for steroid-binding aptamers selected using traditional SELEX approaches. Finally, the selected aptamers were exploited in microtiter plate assays, achieving nanomolar limits of detection, while the specificity of these aptamers was also demonstrated. Overall, the one-pot SELEX strategy led to the discovery of aptamers for three different steroid targets in one single selection without compromising their affinity or specificity, demonstrating the power of this approach of aptamer discovery for the simultaneous selection of aptamers against multiple targets.