+1 (314) 370-6046 or
Contact Us
Submit product reviews and earn rewards >>
Antibodies.com Logo Antibodies.com Logo
Sign In/Register
0

Aptamers

By Ryan Hamnett, PhD

Aptamers are single-stranded DNA or RNA molecules that fold into specific 3D conformations, allowing them to bind with high specificity and affinity to diverse targets such as proteins, small molecules, and cell types (Figure 1).

Diagram of aptamer binding from single stranded RNA or DNA through to target recognition

Figure 1: Aptamer binding. The precise structural conformation of aptamers allows them to bind to targets with high specificity.

Like antibodies, aptamers are easy to label with reporters, enzymes, or fluorescent tags, making them highly applicable to a wide variety of laboratory and diagnostic techniques, including lateral flow assays, biosensing, and histochemistry. Thanks to their ease of production, cost effectiveness, stability and high specificity, aptamers are gaining traction as versatile alternatives to traditional binding molecules such as antibodies.

We offer aptamers against a wide variety of molecules and cell types, including proteins (e.g. receptors, enzymes), hormones (e.g. insulin), metal ions (e.g. lead), nucleosides and nucleotides (e.g. adenosine), viruses (e.g. influenza), bacteria (e.g. E. coli), and disease-related molecules (e.g. alpha-synuclein oligomers).

Explore all Aptamers

Table of Contents

Advantages of Aptamers over Antibodies

Aptamer function is comparable to that of antibodies, binding to specific recognition sites on targets. Similarly to antibodies, aptamers bind with high affinity1 and exhibit high levels of specificity, able to distinguish between proteins that differ by a single amino acid.2 However, aptamers tend to be faster and less expensive to produce, more reproducible between batches (even compared to monoclonal antibodies), and easier to chemically modify.3 Table 1 shows a direct comparison between aptamers and antibodies in a range of key features.

Aptamers Antibodies
Ease of synthesis Chemically synthesized Produced in living systems (animals or cells)
Size and structure Smaller size (~6-30 kDa), better tissue penetration Larger (~150 kDa) and more complex
Temperature stability -80°C – 100°C -80°C – 4°C
pH stability Less sensitive to changes in pH, temperature and ionic conditions More sensitive to environmental conditions
Ease of modification Easily modified or engineered Modification is more complex and may involve genetic engineering
Cost-effectiveness Generally more cost-effective and scalable Can be more expensive and time-consuming
Specificity Highly specific Highly specific
Affinity 10 pM to10 µM 10 pM to10 µM
In vivo tolerance Will not induce immune response. Cleared by renal system without base modification. Degradation by nucleases Can induce immune response via Fc region. Longer circulating half-life

Table 1: Key differences between aptamers and antibodies.

Despite the advantages indicated above, aptamers are still considerably behind antibodies with respect to use in research and clinical settings. For therapeutic use, this may be due to the necessity for aptamer modifications to prevent renal clearance and nuclease degradation.4

Use in laboratories may also be limited by nuclease-mediated degradation, particularly for RNA aptamers, whereas DNases are less likely to be an issue in controlled settings. Instead, an early hurdle was a patent protecting the use of SELEX (systematic evolution of ligands by exponential enrichment), the primary method for developing aptamers that continues to be used today,5 though such patent protection has now expired. SELEX is also labor-intensive, and non-specific background binding during aptamer development significantly affects SELEX efficiency,3,6 but modern innovations in aptamer development are overcoming such issues.7

Finally, as the ‘traditional’ target recognition molecule, antibodies remain at the forefront of biomedical research, with more antibodies currently available for a greater number of target molecules and integrated in research groups’ workflows, despite the plethora of laboratory applications to which aptamers are well-suited.

Aptamer Applications in Biochemical Research

Thanks to the high affinity, specificity, and available modifications of aptamers, aptamers can replace antibodies in many common laboratory techniques with few adjustments to existing workflows, while making use of the unique advantages afforded by aptamers.

Apta-Histochemistry

Tagging aptamers with fluorophores enables them to be used as direct labels in histochemistry, analogous to direct immunolabeling, removing the potential for secondary antibody cross-reactivity. Histochemical signal amplification remains possible through several methods, including tagging the aptamer with an enzyme such as horseradish peroxidase (HRP) instead of a fluorophore, or using biotinylation or enzyme-labelled secondary antibodies directed against the fluorophore on the aptamer.8,9

ELASA

Similar adaptations exist for other biochemical techniques that traditionally rely on antibodies, such as the enzyme-linked immunosorbent assay (ELISA; which becomes enzyme-linked apta-sorbent assay (ELASA)) or western blot.10–14 Like ELISA, ELASA comes in a number of different formats, including direct, indirect, sandwich and competitive (Figure 2). ELASA plates have the potential to be more consistent between batches due to the high reproducibility of aptamers, and ELASA is possible against a wider variety of targets.11 Not only can aptamers be generated more readily for non-immunogenic molecules than antibodies, but aptamers can also recognize environmental pollutants.15 ELASA plates also offer superior reusability potential over ELISA plates, because aptamers can be regenerated.11 Bound antigens can be separated from the aptamer by conditions that would irreparably damage antibodies (heat, acid, salt, proteinase K etc.), but from which aptamers can subsequently refold and be reused.11,16

Diagram of ELASA formats, including schematics of direct, indirect, sandwich and competitive ELASA

Figure 2: ELASA formats. Aptamers in ELASAs are used in a similar way to antibodies in traditional ELISA formats. HRP can be directly conjugated to aptamers17 or linked via biotin-streptavidin.18 Not to scale – aptamers (6-30 kDa) are significantly smaller than antibodies (150 kDa).

Affinity Chromatography

Aptamers are particularly useful in affinity chromatography, where their greater stability can withstand harsh elution conditions.19 Alternatively, targets can be eluted via introduction of the complementary reverse strand, changing the conformation of the aptamer and so eluting the target under very mild conditions to preserve target integrity.20 The smaller size of aptamers allows a greater density of aptamers to be immobilized to solid substrates, increasing potential binding capacity, and immobilization of aptamers is easier than antibodies due to aptamers being more amenable to chemical modification.

Apta-Precipitation

The aptamer equivalent of immunoprecipitation, apta-precipitation,21 can be used to precipitate not only proteins, but also small molecules such as adenosine,22 and even cell populations23 in a technique akin to immunopanning.24 Apta-precipitation suffers from fewer contaminants being carried over to downstream mass spectrometry analysis than traditional immunoprecipitation, because off-target binding is more likely to occur on antibodies than aptamers.25

Aptamers as Biosensors

Biosensors use a biological component to detect the presence of an analyte and output a signal proportional to the analyte’s concentration. Biosensors often produce real-time results, tend to be inexpensive, and do not require specialized equipment to operate, making them highly accessible for point-of-care diagnostics and environmental monitoring. The properties of aptamers as recognition elements, including their stability, cost, and chemical versatility, make them strong candidates for biosensors.3,26–28

The most common forms of biosensor output come in the form of electrical signal, color, or fluorescence. Colorimetric biosensors in particular are useful in diagnostics, using a color change visible to the naked eye to indicate the presence of a target. The lateral flow assay (LFA) is a well-known colorimetric biosensor, widely used in pregnancy tests and COVID-19 rapid antigen tests. LFAs rely on a sample being transported by capillary action up an absorbent strip, on which it binds to a binding molecule (e.g. antibodies or aptamer) conjugated to nanoparticles. A visible colored line appears at the site of nanoparticle accumulation at the positive test line and/or the control line, with accumulation occurring due to the presence of additional binding molecules at those sites (Figure 3). Chemical modification of aptamers makes them easier to conjugate to nanoparticles with a consistent orientation than antibodies, and they also have a longer shelf-life at room temperature.

Diagram of how a lateral flow assay works

Figure 3: Lateral Flow Assay using aptamers. Sample containing analyte is dropped onto the sample pad, and then flows by capillary action. Analyte specifically binds to the nanoparticle-conjugated aptamer, which is then carried to the test and control lines. Nanoparticles bound to analyte are captured by the detection probe and accumulate at the test line, while excess nanoparticle-aptamer conjugates carry on to the control probe to confirm the test is working. The control probe may be streptavidin, to capture a biotinylated aptamer, or it may be a complementary oligomer.29 Accumulation of gold nanoparticles produce visible red or blue lines depending on nanoparticle size.30

Clinical Applications of Aptamers

Aptamers have the potential for extensive use in clinical settings, with the first therapeutic aptamer, pegaptanib (Macugen), approved by the US FDA in 2004.31 Aptamers are able to bind target molecules and deliver therapeutic compounds with similar alacrity to antibodies, while being cheaper to produce and maintain. This makes them particularly attractive for point of care testing,32 but they have also found applications in diagnostics,33,34 drug discovery,4,35 and treatment.36

How Aptamers are Developed

Aptamers are traditionally developed by SELEX, which was conceived in 199037 and continues to be used today, although new methods are being developed to overcome issues with SELEX, such as being labor-intensive and sometimes failing to produce aptamers of sufficiently high selectivity.7 SELEX involves repeated rounds of exposing an oligonucleotide library (the putative aptamers) to the target, identifying and isolating the oligonucleotides with the strongest target binding properties, amplifying the selected nucleotides, and repeating the process under more stringent conditions (Figure 4). This cycle is usually repeated 10-20 times until an aptamer of sufficient affinity and selectivity is found.

Diagram of how SELEX works to generate specific aptamers

Figure 4: SELEX process for producing aptamers.

Aptamers are usually made of single stranded DNA or RNA, though non-natural nucleic acids and mirrored enantiomers (L-DNA or L-RNA) have also been used, increasing aptamer diversity and stability, respectively.3 While no differences have been observed between DNA and RNA in terms of specificity or affinity, DNA tends to be more stable, cheaper, and does not require reverse transcription, while RNA is more flexible and so can fold into a greater number of potential structures.11 Most recently discovered and applied aptamers are DNA-based.3

A library of 1014–1015 unique, random aptamers is used as a starting library. Each aptamer contains a random region for binding, flanked by two constant 5’ and 3’ regions for PCR amplification and, in some cases, structure. The length of each aptamer varies between experiments, but random regions are typically 36-70 nt long.3,38 RNA libraries are generated by in vitro transcription of DNA templates, while DNA libraries must be prepared by strand separation of double stranded PCR products.4

Once generated, the sequences are exposed to the target (protein, metal ion, small molecule etc.), which has often been immobilized to a solid substrate such as a bead for manipulation.39 Aptamers that bind successfully are retained by the bead-bound target, while non-binding aptamers are washed away. The bound sequences are eluted away from the target and amplified by PCR to create another library. Subsequent rounds of selection are performed under different stringency conditions to ultimately identify sequences with the strongest binding properties.39

Explore all Aptamers

References

Diagrams created with BioRender.com.

  1. Alves Ferreira-Bravo, I., Cozens, C., Holliger, P. & DeStefano, J. J. Selection of 2′-deoxy-2′-fluoroarabinonucleotide (FANA) aptamers that bind HIV-1 reverse transcriptase with picomolar affinity. Nucleic Acids Res. 43, 9587–9599 (2015).
  2. Chen, L. et al. The isolation of an RNA aptamer targeting to p53 protein with single amino acid mutation. Proc. Natl. Acad. Sci. 112, 10002–10007 (2015).
  3. Yang, L. F., Ling, M., Kacherovsky, N. & Pun, S. H. Aptamers 101: aptamer discovery and in vitro applications in biosensors and separations. Chem. Sci. 14, 4961–4978 (2023).
  4. Keefe, A. D., Pai, S. & Ellington, A. Aptamers as therapeutics. Nat. Rev. Drug Discov. 9, 537–550 (2010).
  5. Sampson, T. Protecting intellectual property rights in SELEX and aptamers. World Pat. Inf. 25, 343–349 (2003).
  6. Wang, J., Rudzinski, J. F., Gong, Q., Soh, H. T. & Atzberger, P. J. Influence of Target Concentration and Background Binding on In Vitro Selection of Affinity Reagents. PLOS ONE 7, e43940 (2012).
  7. Gotrik, M. R., Feagin, T. A., Csordas, A. T., Nakamoto, M. A. & Soh, H. T. Advancements in Aptamer Discovery Technologies. Acc. Chem. Res. 49, 1903–1910 (2016).
  8. Murakami, K. et al. An RNA aptamer with potent affinity for a toxic dimer of amyloid β42 has potential utility for histochemical studies of Alzheimer’s disease. J. Biol. Chem. 295, 4870–4880 (2020).
  9. Liu, M. et al. A novel aptamer-based histochemistry assay for specific diagnosis of clinical breast cancer tissues. Chin. Chem. Lett. 32, 1726–1730 (2021).
  10. Shin, S., Kim, I.-H., Kang, W., Yang, J. K. & Hah, S. S. An alternative to Western blot analysis using RNA aptamer-functionalized quantum dots. Bioorg. Med. Chem. Lett. 20, 3322–3325 (2010).
  11. Toh, S. Y., Citartan, M., Gopinath, S. C. B. & Tang, T.-H. Aptamers as a replacement for antibodies in enzyme-linked immunosorbent assay. Biosens. Bioelectron. 64, 392–403 (2015).
  12. Lee, K. H. & Zeng, H. Aptamer-Based ELISA Assay for Highly Specific and Sensitive Detection of Zika NS1 Protein. Anal. Chem. 89, 12743–12748 (2017).
  13. Wang, Y., Li, Z. & Yu, H. Aptamer-Based Western Blot for Selective Protein Recognition. Front. Chem. 8, (2020).
  14. Zhang, X. et al. A Novel Sandwich ELASA Based on Aptamer for Detection of Largemouth Bass Virus (LMBV). Viruses 14, 945 (2022).
  15. Hayat, A. & Marty, J. L. Aptamer based electrochemical sensors for emerging environmental pollutants. Front. Chem. 2, (2014).
  16. Wu, Z.-S. et al. Reusable Electrochemical Sensing Platform for Highly Sensitive Detection of Small Molecules Based on Structure-Switching Signaling Aptamers. Anal. Chem. 79, 2933–2939 (2007).
  17. Wu, S.-W. et al. Novel enzyme-linked aptamer-antibody sandwich assay and hybrid lateral flow strip for SARS-CoV-2 detection. J. Nanobiotechnology 22, 5 (2024).
  18. Vargas-Montes, M. et al. Enzyme-Linked Aptamer Assay (ELAA) for Detection of Toxoplasma ROP18 Protein in Human Serum. Front. Cell. Infect. Microbiol. 9, (2019).
  19. Zhao, Q., Wu, M., Chris Le, X. & Li, X.-F. Applications of aptamer affinity chromatography. TrAC Trends Anal. Chem. 41, 46–57 (2012).
  20. Gray, B. P., Requena, M. D., Nichols, M. D. & Sullenger, B. A. Aptamers as Reversible Sorting Ligands for Preparation of Cells in Their Native State. Cell Chem. Biol. 27, 232-244.e7 (2020).
  21. Fayazi, R., Habibi-Rezaei, M., Heiat, M., Javadi-Zarnaghi, F. & Taheri, R. A. Glycated albumin precipitation using aptamer conjugated magnetic nanoparticles. Sci. Rep. 10, 10716 (2020).
  22. Deng, Q., German, I., Buchanan, D. & Kennedy, R. T. Retention and Separation of Adenosine and Analogues by Affinity Chromatography with an Aptamer Stationary Phase. Anal. Chem. 73, 5415–5421 (2001).
  23. Phillips, J. A., Xu, Y., Xia, Z., Fan, Z. H. & Tan, W. Enrichment of Cancer Cells Using Aptamers Immobilized on a Microfluidic Channel. Anal. Chem. 81, 1033–1039 (2009).
  24. Barres, B. A. Designing and Troubleshooting Immunopanning Protocols for Purifying Neural Cells. Cold Spring Harb. Protoc. 2014, pdb.ip073999 (2014).
  25. Ray, J. et al. RNA aptamer capture of macromolecular complexes for mass spectrometry analysis. Nucleic Acids Res. 48, e90 (2020).
  26. Tombelli, S., Minunni, M. & Mascini, M. Analytical applications of aptamers. Biosens. Bioelectron. 20, 2424–2434 (2005).
  27. Zhang, Y., Lai, B. S. & Juhas, M. Recent Advances in Aptamer Discovery and Applications. Molecules 24, 941 (2019).
  28. Khan, N. I. & Song, E. Lab-on-a-Chip Systems for Aptamer-Based Biosensing. Micromachines 11, 220 (2020).
  29. Huang, L. et al. Aptamer-based lateral flow assay on-site biosensors. Biosens. Bioelectron. 186, 113279 (2021).
  30. Ardekani, L. S. & Thulstrup, P. W. Gold Nanoparticle-Mediated Lateral Flow Assays for Detection of Host Antibodies and COVID-19 Proteins. Nanomaterials 12, 1456 (2022).
  31. Xiao, X., Li, H., Zhao, L., Zhang, Y. & Liu, Z. Oligonucleotide aptamers: Recent advances in their screening, molecular conformation and therapeutic applications. Biomed. Pharmacother. 143, 112232 (2021).
  32. Zhao, L. et al. Aptamer-based point-of-care-testing for small molecule targets: From aptamers to aptasensors, devices and applications. TrAC Trends Anal. Chem. 169, 117408 (2023).
  33. Jayasena, S. D. Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics. Clin. Chem. 45, 1628–1650 (1999).
  34. Thiviyanathan, V. & Gorenstein, D. G. Aptamers and the Next Generation of Diagnostic Reagents. Proteomics Clin. Appl. 6, 563–573 (2012).
  35. Chandola, C., Kalme, S., Casteleijn, M. G., Urtti, A. & Neerathilingam, M. Application of aptamers in diagnostics, drug-delivery and imaging. J. Biosci. 41, 535–561 (2016).
  36. Agnello, L., Camorani, S., Fedele, M. & Cerchia, L. Aptamers and antibodies: rivals or allies in cancer targeted therapy? Explor. Target. Anti-Tumor Ther. 2, 107–121 (2021).
  37. Tuerk, C. & Gold, L. Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase. Science 249, 505–510 (1990).
  38. Legiewicz, M., Lozupone, C., Knight, R. & Yarus, M. Size, constant sequences, and optimal selection. RNA 11, 1701–1709 (2005).
  39. Kohlberger, M. & Gadermaier, G. SELEX: Critical factors and optimization strategies for successful aptamer selection. Biotechnol. Appl. Biochem. 69, 1771–1792 (2022).
Show all