Here, we demonstrate that the use of the single-molecule centroid localization algorithm can improve the accuracy of fluorescence binding assays. Two major artifacts in this type of assay, i.e., nonspecific binding events and optically overlapping receptors, can be detected and corrected during analysis. The effectiveness of our method was confirmed by measuring two weak biomolecular interactions, the interaction between the B1 domain of streptococcal protein G and immunoglobulin G and the interaction between double-stranded DNA and the Cas9–RNA complex with limited sequence matches. This analysis routine requires little modification to common experimental protocols, making it readily applicable to existing data and future experiments.

中文翻译：

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单分子质心定位算法提高了荧光结合测定的准确性

在这里，我们证明了使用单分子质心定位算法可以提高荧光结合测定的准确性。可以在分析过程中检测和纠正这种类型的分析中的两个主要伪影，即非特异性结合事件和光学重叠的受体。通过测量两个弱的生物分子相互作用，即链球菌蛋白G的B1结构域和免疫球蛋白G之间的相互作用，以及双链DNA和有限序列匹配的Cas9-RNA复合物之间的相互作用，我们的方法的有效性得到了证实。该分析程序几乎不需要修改常见的实验方案，因此很容易应用于现有数据和将来的实验。