Hua Boyang, Wang Yanbo, Park Seongjin, Han Kyu Young, Singh Digvijay, Kim Jin H, Cheng Wei, Ha Taekjip
Department of Biophysics and Biophysical Chemistry , Johns Hopkins School of Medicine , Baltimore , Maryland 21205 , United States.
Department of Physics , University of Illinois at Urbana-Champaign , Urbana , Illinois 61801 , United States.
Biochemistry. 2018 Mar 13;57(10):1572-1576. doi: 10.1021/acs.biochem.7b01293. Epub 2018 Feb 28.
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.
在此，我们证明使用单分子质心定位算法可提高荧光结合测定的准确性。在这类测定中存在的两个主要伪影，即非特异性结合事件和光学重叠受体，在分析过程中可被检测并校正。通过测量两种弱生物分子相互作用，即链球菌蛋白G的B1结构域与免疫球蛋白G之间的相互作用以及双链DNA与具有有限序列匹配的Cas9-RNA复合物之间的相互作用，证实了我们方法的有效性。这种分析程序对常见实验方案几乎无需修改，使其易于应用于现有数据和未来实验。
