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Overcoming Effects of Heterogeneous Binding on BLI Analysis.

Noah Sherer1, Jae-Hyun Cho1

  • 1Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, United States.

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Analyte aggregation on biosensors causes signal drift in biolayer interferometry (BLI) analysis, leading to inaccurate binding characteristics. This study identifies aggregation as a source of heterogeneous binding and proposes mitigation strategies for reliable biomolecular interaction studies.

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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.