Biophysical Characterization of Aptamer-Target Interactions

  • Maximilian Plach
  • Thomas SchubertEmail author
Part of the Advances in Biochemical Engineering/Biotechnology book series (ABE, volume 174)


Aptamers are single-stranded nucleic acid molecules forming well-defined 3D structures. Aptamers typically bind to their ligands with high affinity and specificity. They are capable of interacting with various kinds of ligands: ions, small molecules, peptides, proteins, viruses, bacteria, and even cells. Therefore, aptamers are in widespread use as sensor molecules or as targeting agents in diagnostics and pharmaceutics. As a prerequisite for their use in these economic high-value areas, aptamers must be studied in detail with respect to different biophysical characteristics. Of central importance are basic binding parameters of the aptamer-target interaction, such as binding affinity and kinetics. Numerous biophysical methods with different features, characteristics, and capabilities are used in the field today for this purpose.

This chapter provides an overview of the current state-of-the-art technologies for studying interactions between aptamers and targets and discusses their advantages as well as drawbacks. Furthermore, essential aspects influencing any aptamer characterization strategy will be presented. Finally, issues of comparability of binding data between different aptamer characterization technologies will be discussed.

Graphical Abstract


Affinity Binding parameters Biolayer Interferometry Biophysical characterization EMSA Filter-Binding Assay Flow Cytometry Fluorescence Polarization Isothermal Titration Calorimetry Kinetics MicroScale Thermophoresis Surface Plasmon Resonance Thermodynamics SwitchSense 


  1. 1.
    Chang AL, McKeague M, Liang JC, Smolke CD (2014) Kinetic and equilibrium binding characterization of aptamers to small molecules using a label-free, sensitive, and scalable platform. Anal Chem 86(7):3273–3278CrossRefGoogle Scholar
  2. 2.
    Amano R, Takada K, Tanaka Y, Nakamura Y, Kawai G, Kozu T, Sakamoto T (2016) Kinetic and thermodynamic analyses of interaction between a high-affinity RNA aptamer and its target protein. Biochemistry 55(45):6221–6229CrossRefGoogle Scholar
  3. 3.
    Stoltenburg R, Schubert T, Strehlitz B (2015) In vitro selection and interaction studies of a DNA aptamer targeting protein A. PLoS One 10:e0134403. Scholar
  4. 4.
    Fülle L et al (2018) RNA aptamers recognizing murine CCL17 inhibit T cell chemotaxis and reduce contact hypersensitivity in vivo. Mol Ther 26(1):95–104. Scholar
  5. 5.
    Wochner A, Menger M, Orgel D, Cech B, Rimmele M, Erdmann VA, Glökler J (2008) A DNA aptamer with high affinity and specificity for therapeutic anthracyclines. Anal Biochem 373(1):34–42CrossRefGoogle Scholar
  6. 6.
    Lou X, Egli M, Yang X (2016) Determining functional aptamer-protein interaction by biolayer interferometry. Curr Protoc Nucleic Acid Chem 67:7.25.1–7.25.15. Scholar
  7. 7.
    Espiritu CAL, Justo CAC, Rubio MJ, Svobodova M, Bashammakh AS, Alyoubi AO, Rivera WL, Rollon AP, O’Sullivan CK (2018) Aptamer selection against a trichomonas vaginalis adhesion protein for diagnostic applications. ACS Infect Dis 4(9):1306–1315. Scholar
  8. 8.
    Sakamoto T, Ennifar E, Nakamura Y (2018) Thermodynamic study of aptamers binding to their target proteins. Biochimie 145:91–97. Scholar
  9. 9.
    Poongavanam MV, Kisley L, Kourentzi K, Landes CF, Willson RC (2016) Ensemble and single-molecule biophysical characterization of D17.4 DNA aptamer-IgE interactions. Biochim Biophys Acta 1864(1):154–164. Scholar
  10. 10.
    Geng X et al (2013) Screening interaction between ochratoxin A and aptamers by fluorescence anisotropy approach. Anal Bioanal Chem 405(8):2443–2449. Scholar
  11. 11.
    Sefah K, Shangguan D, Xiong X, O’Donoghue MB, Tan W (2010) Development of DNA aptamers using cell-SELEX. Nat Protoc 5(6):1169–1185CrossRefGoogle Scholar
  12. 12.
    Soundy J, Day D (2017) Selection of DNA aptamers specific for live Pseudomonas aeruginosa. PLoS One 12(9):e0185385. Scholar
  13. 13.
    Jauset Rubio M, Svobodová M, Mairal T, Schubert T, Künne S, Mayer G, O’Sullivan CK (2016) β-Conglutin dual aptamers binding distinct aptatopes. Anal Bioanal Chem 408(3):875–884. Scholar
  14. 14.
    Shiohara T, Saito H, Inoue T (2009) A designed RNA selection: establishment of a stable complex between a target and selectant RNA via two coordinated interaction. Nucleic Acids Res 37(3):e23. Scholar
  15. 15.
    Lönne M, Bolten S, Lavrentieva A, Stahl F, Scheper T, Walter J-G (2015) Development of an aptamer-based affinity purification method for vascular endothelial growth factor. Biotechnol Rep 8:16–23. Scholar
  16. 16.
    Krepl M, Blatter M, Cléry A, Damberger FF, Allain FHT, Sponer J (2017) Structural study of the Fox-1 RRM protein hydration reveals a role for key water molecules in RRM-RNA recognition. Nucleic Acids Res 45(13):8046–8063. Scholar
  17. 17.
    Skouridou V, Schubert T, Bashammakh AS, El-Shahawi MS, Alyoubi AO, O’Sullivan CK (2017) Aptatope mapping of the binding site of a progesterone aptamer on the steroid ring structure. Anal Biochem 531:8–11CrossRefGoogle Scholar
  18. 18.
    Zihlmann P, Silbermann M, Sharpe T, Jiang X, Mühlethaler T, Jakob RP, Rabbani S, Sager CP, Frei P, Pang L, Maier T, Ernst B (2018) KinITC-one method supports both thermodynamic and kinetic SARs as exemplified on FimH antagonists. Chemistry 24(49):13049–13057. Scholar
  19. 19.
    de Mol NJ, Dekker FJ, Broutin I, Fischer MJ, Liskamp RM (2005) Surface plasmon resonance thermodynamic and kinetic analysis as a strategic tool in drug design. Distinct ways for phosphopeptides to plug into Src- and Grb2 SH2 domains. J Med Chem 48(3):753–763CrossRefGoogle Scholar
  20. 20.
    Bastian M, Heymann S, Jacomy M (2009) Gephi: an open source software for exploring and manipulating networks. In: International AAAI conference on web and social mediaGoogle Scholar

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© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.2bind GmbHRegensburgGermany

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