Sherer Noah, Cho Jae-Hyun
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, United States.
ACS Omega. 2025 Jun 25;10(26):28422-28428. doi: 10.1021/acsomega.5c03942. eCollection 2025 Jul 8.
Binding characteristics, such as , , and , are critical for the mechanistic study of biomolecular interactions and drug design. Biolayer interferometry (BLI) has become popular due to its simplicity and sensitivity. Despite its widespread use, BLI data analysis is susceptible to various non-ideal features in sensorgrams. One commonly observed issue is a persistent signal drift after the binding process reaches an expected steady state. The basis of this phenomenon, often referred to as heterogeneous binding, remains poorly understood. In this study, we find that analyte aggregation on the biosensor, particularly induced by ligand-analyte complexes, can contribute to heterogeneous binding. We also find that heterogeneous binding affects not only the association phase but also the dissociation process, leading to erroneous binding characteristics. We propose an approach to mitigate the adverse impacts of heterogeneous binding on the BLI analysis. Since accurate binding characterization is fundamental for many biophysical analyses, addressing this issue is crucial.
诸如亲和力、结合速率和解离速率等结合特性，对于生物分子相互作用的机理研究和药物设计至关重要。生物层干涉术（BLI）因其操作简便和灵敏度高而受到广泛应用。尽管BLI已被广泛使用，但其数据分析易受传感图中各种非理想特征的影响。一个常见的问题是在结合过程达到预期稳态后出现持续的信号漂移。这种现象（通常称为异质性结合）的原因仍知之甚少。在本研究中，我们发现生物传感器上的分析物聚集，特别是由配体 - 分析物复合物诱导的聚集，会导致异质性结合。我们还发现异质性结合不仅影响结合阶段，还影响解离过程，从而导致错误的结合特性。我们提出了一种方法来减轻异质性结合对BLI分析的不利影响。由于准确的结合表征是许多生物物理分析的基础，解决这个问题至关重要。
