Binding Affinity and Stoichiometry of a Multivalent Protein-aptamer Association

Importance of the topic

A detailed understanding of multivalent biomolecular interactions is critical for the development and optimization of therapeutic agents, including monoclonal antibodies, peptides and aptamers. Precise measurement of binding affinity and absolute stoichiometry under native conditions enables better prediction of efficacy, selectivity and mechanism of action in pharmaceutical research and industrial quality control.

Objectives and study overview

This study applied composition-gradient multi-angle light scattering to quantify the equilibrium affinity and stoichiometry of a DNA aptamer binding to the tetrameric protein streptavidin. The goal was to determine number of binding sites and the dissociation constant in solution without labels or surfaces that could perturb the interaction.

Methodology

The approach relies on generating concentration gradients of each component and mixed solutions via an automated gradient system. Light scattering and concentration data are collected at equilibrium and fitted to association models to extract weight-average molar mass and equilibrium constants. This allows identification of specific complexes and their stoichiometry.

Used Instrumentation

• Calypso II automated composition gradient delivery
• HELEOS II multi-angle light scattering detector
• Dual-wavelength UV concentration detector

Main results and discussion

Neither streptavidin nor the DNA aptamer self-associate over the tested concentration range. Mixed solutions show excess light scattering indicative of complex formation. Data fitting reveals two independent aptamer-binding sites per streptavidin molecule, contrary to the expected four. The dissociation constant at each site is KD = 190 nM, consistent with other techniques. No cooperativity or higher-order complexes such as 1:3 or 1:4 are observed, suggesting streptavidin functions as a dimer of dimers under these conditions.

Benefits and practical applications of the method

Composition-gradient MALS offers absolute measurement of binding stoichiometry and affinity in native solution without labels or immobilization. This is valuable for characterizing complex therapeutic candidates, verifying molecular interaction models, and supporting quality control in drug development and production.

Future trends and potential applications

Further applications may include studies of bispecific antibodies, virus-like particles and other multivalent systems. Integration with complementary biophysical techniques and high-throughput gradient automation could expand its use in early discovery, formulation screening and structural biochemistry.

Conclusion

CG-MALS provides a rigorous, label-free approach to quantify binding affinity and absolute stoichiometry of macromolecular interactions in solution. The unexpected 1:2 aptamer:streptavidin stoichiometry underscores the method’s power to reveal biologically relevant binding architectures that may be missed by surface-based assays.

References

• Bing T; et al. Bioorg Med Chem 2010;18:1798.
• Ruigrok VJ; et al. ChemBioChem 2012;13:829.