nucleic acid aptamers are artificial single-stranded oligomers capable of specifically binding to a particular target molecule or target family. They can be composed of DNA, RNA, XNA, or peptide nucleic acids and achieve molecular recognition through unique three-dimensional conformations. With binding affinities typically ranging from the picomolar to micromolar scale, aptamers can bind with antibody-like selectivity, earning them the nickname "chemical antibodies." Compared to conventional antibodies, aptamers offer key advantages such as chemical synthesis feasibility, low immunogenicity, small molecular size, and ease of modification. Because they can target proteins, small molecules, metal ions, and even whole cells, aptamers have emerged as essential molecular tools in precision medicine, biomarker discovery, and targeted delivery.

Figure 1. Schematic representation of the functionality of aptamers. (Stoltenburg R, et al., 2007)

Over the past two decades, aptamer research has transitioned from theoretical exploration to diverse applications. Early studies focused primarily on molecular recognition mechanisms and screening methodologies. More recently, as targeted therapy, imaging, and nanotechnology have rapidly advanced, aptamers—being programmable nucleic acid structures—have found broad potential in drug design, delivery systems, and disease diagnostics. Unlike protein antibodies, aptamers can be entirely synthesized chemically, without reliance on animal immune systems, allowing precise control of structure and purity. This confers unique advantages in terms of quality consistency and manufacturing scalability.

SELEX Technology: The Core of Aptamer Discovery

The identification of aptamers relies on an in vitro selection technique known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Through iterative rounds of binding, elution, and amplification within a randomized oligonucleotide library, SELEX enriches sequences that exhibit high target affinity. The canonical SELEX workflow consists of library generation, target incubation, separation of bound sequences, amplification, and regeneration of single-stranded DNA.

The process begins with a chemically synthesized oligonucleotide library containing a random region (typically 20–60 nucleotides) flanked by fixed primer sites. Following PCR amplification, the library is incubated with an immobilized target under carefully optimized temperature, time, ionic strength, and buffer conditions that promote stable nucleic acid folding into secondary or tertiary structures for efficient target recognition. Bound sequences are eluted, amplified, and subjected to subsequent selection rounds. As iterations proceed, the library becomes enriched in high-affinity, high-specificity sequences.

A key to SELEX success lies in fine-tuning selection pressure. The introduction of counter-selection significantly improves specificity by pre-incubating the library with non-target or structurally similar molecules to remove nonspecific binders. Traditional SELEX may require over ten rounds to obtain ideal aptamers, but advances in automation, microfluidics, and high-throughput sequencing have drastically reduced the number of rounds and overall time required.

Figure 2. Traditional SELEX Workflow. (Wang, et al., 2023)

Evolution and Diversification of SELEX Platforms

As research progressed, SELEX technology evolved from classical methods to diversified, specialized platforms. Early nitrocellulose filtration-based SELEX enabled the first aptamer discoveries but suffered from issues like unstable binding and low elution efficiency. Affinity chromatography SELEX, utilizing immobilized beads, improved target control yet remained limited by steric hindrance and nonspecific adsorption.

To address these challenges, capillary electrophoresis SELEX (CE-SELEX) was introduced. By exploiting differences in free electrophoretic mobility between complexes and unbound oligonucleotides, CE-SELEX significantly increased screening speed and resolution, requiring only a few rounds to identify high-affinity sequences. However, CE-SELEX is sensitive to sample concentration and injection conditions, often necessitating microfluidic free-flow electrophoresis optimization.

Magnetic bead SELEX (Mag-SELEX) employs magnetic nanoparticles as solid-phase supports, allowing efficient magnetic separation of bound and unbound sequences, thereby improving reproducibility and throughput—especially for small-molecule or low-abundance targets. Building upon this, Cell-SELEX uses whole cells as targets, enabling the selection of aptamers that recognize native membrane proteins or unknown biomarkers. It has become a cornerstone for tumor-specific aptamer discovery. Coupled with fluorescence-activated cell sorting (FACS) and high-throughput sequencing, Cell-SELEX accelerates screening while eliminating sequences that bind normal cells through counter-selection, yielding clinically valuable aptamer candidates.

Figure 3. Magnetic Bead SELEX. (Harbaugh SV, et al., 2018)

Figure 4. Cell-SELEX. (Wang, et al., 2023)

The emergence of microfluidic SELEX marked a leap toward automation and integration. By miniaturizing incubation, separation, elution, and amplification steps within a single microchip, this system enables continuous, low-volume SELEX operations, reducing sample use and human error.

Figure 5. Microfluidic SELEX. (Dembowski SK, et al., 2017.)

Meanwhile, in vivo SELEX extends screening into physiologically relevant settings. Conducted directly within animal models, it identifies aptamers capable of recognizing targets in native biological environments—a powerful strategy for tumor targeting and drug delivery studies.

In addition, cross-SELEX, mirror-image SELEX, truncated SELEX, and chimeric SELEX approaches enhance success rates and molecular stability through multi-target or structure-optimization strategies. Collectively, these innovations have greatly expanded the specificity, affinity, and application spectrum of aptamers.

Figure 6. Cross-SELEX. (Wang, et al., 2023)

Chemical Modification and Functionalization of Aptamers

While natural aptamers exhibit strong binding affinities, their in vivo applications are limited by nuclease degradation and rapid renal clearance. Various chemical modification strategies have been developed to improve pharmacokinetics. For example, introducing 2'-fluoro, 2'-O-methyl, or 2'-amino groups on the ribose enhances nuclease resistance. Conjugation with macromolecules such as PEG, liposomes, or proteins extends circulation time and reduces renal filtration. Additional approaches like end-capping, hydrophobic chain extension, and steric shielding can further improve stability without compromising binding performance.

Beyond stabilization, aptamers serve as modular components in multifunctional molecular systems. Conjugation with fluorescent dyes, quantum dots, or gold nanoparticles yields ultrasensitive molecular probes. Linking aptamers with drugs, siRNA, or CRISPR components enables targeted drug delivery or gene editing. Integration into DNA nanostructures or circular RNA frameworks enhances conformational stability and multivalent binding. These chemical and structural modifications lay a robust foundation for multidimensional aptamer applications.

Therapeutic and Diagnostic Applications of Aptamers

Aptamers demonstrate therapeutic and diagnostic potential comparable to, and in some cases surpassing, antibodies. They can inhibit disease progression by blocking protein–protein interactions, ligand–receptor binding, or signaling pathways. The anti-VEGF aptamer Pegaptanib (Macugen), the first FDA-approved aptamer drug, validated the clinical feasibility of aptamers in treating age-related macular degeneration. Other aptamer candidates targeting thrombosis, tumor growth, and viral infections are in various clinical stages.

In oncology, aptamers specifically recognize tumor-associated surface molecules such as PSMA, MUC1, and VEGF, thereby enabling targeted blockade or cytotoxic drug delivery. When integrated with nanocarriers, aptamers can form targeted drug delivery systems that enhance tumor accumulation and reduce systemic toxicity. In antiviral research, RNA aptamers binding viral surface proteins or replication enzymes can inhibit infection or replication, offering promising antiviral therapeutic strategies.

In diagnostics, aptamers' stability and specificity have led to their extensive use in biosensor development. Aptasensors, employing electrochemical, fluorescent, or surface plasmon resonance transduction mechanisms, allow ultrasensitive detection of disease biomarkers, toxins, and pollutants. Compared to antibody-based probes, aptamer sensors offer greater reproducibility and storage stability, making them ideal for portable and on-site diagnostics.

Figure 7. Diagnostic and Therapeutic Aptamers in Clinical Trials. (Di Mauro V, et al., 2023)

The convergence of aptamer technology with other therapeutic platforms will also define future innovation. Hybrid systems combining aptamers with antibodies, peptides, or small molecules may offer multivalent and targeted delivery capabilities. In synergy with CRISPR, mRNA, or siRNA therapeutics, aptamers can achieve higher precision in gene targeting and regulation. Research on aptamer–nanocomposites, photosensitive aptamers, and dynamically controllable aptamer systems is paving the way toward intelligent therapeutics and precision medicine.

Conclusion

In summary, nucleic acid aptamers—owing to their exceptional molecular recognition, structural flexibility, and chemical tunability—have become vital connectors between chemistry and biomedicine. From classical SELEX to intelligent and automated selection systems, aptamer technology continues to advance across drug development, molecular diagnostics, and biosensing. Building on this progress, Creative Biogene offers integrated https://www.creative-biogene.com/Services/Aptamers/Aptamers-Services.html, covering SELEX library design, high-throughput screening, and chemical modification, helping researchers accelerate the translation of aptamer technologies into innovative diagnostic and therapeutic applications.