Overcoming Effects of Heterogeneous Binding on BLI Analysis

Area of Science:

• Biophysics
• Biochemistry
• Analytical Chemistry

Background:

• Binding characteristics (k_on, k_off, K_D) are vital for studying biomolecular interactions and drug design.
• Biolayer interferometry (BLI) is a sensitive and simple technique for measuring these interactions.
• BLI data analysis can be affected by non-ideal sensorgram features, including persistent signal drift.

Purpose of the Study:

• To investigate the underlying causes of heterogeneous binding observed in BLI.
• To understand how heterogeneous binding impacts the accuracy of binding characteristic measurements.
• To propose methods for mitigating the adverse effects of heterogeneous binding on BLI data analysis.

Main Methods:

• Utilized biolayer interferometry (BLI) to monitor biomolecular interactions.
• Analyzed sensorgram data to identify and characterize signal drift.
• Investigated the role of analyte aggregation on biosensors in causing heterogeneous binding.

Main Results:

• Analyte aggregation on the biosensor, especially from ligand-analyte complexes, contributes to heterogeneous binding.
• Heterogeneous binding affects both association and dissociation phases, leading to erroneous binding kinetics (k_on, k_off) and affinity (K_D).
• Identified specific conditions that promote analyte aggregation and subsequent signal drift.

Conclusions:

• Analyte aggregation is a significant contributor to heterogeneous binding in BLI.
• Accurate binding characterization requires addressing heterogeneous binding to avoid misleading results.
• The proposed approach can improve the reliability of BLI for biophysical analyses and drug discovery.