## 4 Discussion  
Here, we show the interaction between ACE2 and spike protein 1 using FCCS. Our results indicate that FCCS is a powerful tool to directly and quantitatively determine the interaction between a spike protein of SARS-CoV-2 and its human receptor ACE2 in a solution that does not depend on solid-liquid phase-based assays (e.g., pull-down  
Figure 3: Interaction analysis between ACE2 and S1 subunit using fluorescence cross-correlation spectroscopy (FCCS). (**A**) Typical normalized cross-correlation functions (nCCFs: [\(G_{\text{c}}\)(r)-1/\(G_{\text{r}}\)(0)-1] of mixtures of purified mCherry monomers (Ctrl; grey), ER-mCherry-S1 (S1; green), or ER-mCherry-S1-2 (S1-2; magenta) with hACE2+eGFP. The _x_-axis shows time (r). (**B**) Relative cross-correlation amplitudes (RCAs) of mixtures of two fluorescent molecules. Mo indicates GFP or mCherry monomers. Bars indicate mean \(\pm\) SE. Dots indicate independent values. Statistics: *** \(p<0.001\), NS: \(p\geq 0.05\) (not significant); comparison against the mixture including hACE2-eGFP and mCherry monomers as a negative control or line-indicated pairs. (**C**) Counts per molecule (CPM) of eGFP-tagged proteins during FCCS measurement. (**D**) Counts per molecule (CPM) of mCherry-tagged proteins during FCCS measurement. assay and SPR). Solution-based measurements and the short measurement time (less than 1 min per \(\sim\)10 uL sample) of FCS can be applied for the high-throughput screening of inhibitory drugs and neutralization antibodies, determining any interactions between them. This could lead to the prevention of virus infection and reduce severity where infection does occur. In this report, affinity purification for a polyhistidine tag was performed, but highly purified samples could not be obtained. This is because the intracellular expression of fluorescent protein-tagged hACE2, S1, and S1-2 was low, and some proteins bound nonspecifically to the carrier for affinity purification. To obtain highly purified samples, additional affinity purification and/or other column chromatography techniques (e.g., gel filtration), in addition to an increased number of cells, could be used. However, highly purified samples were not required for interaction analysis using FCCS because FCCS is a fluorescence-based detection of protein interaction with a high target specificity [22]. CPMs of GFP-tagged ACE2 and mCherry-tagged S1 or S1-2 were not different from those of highly purified monomeric GFP and mCherry (Figure 3C,D), indicating that fluorescence brightness was not affected by the protein impurities. Moreover, since RCA shows relative interaction strength, the effect of the interaction inhibitors can be effectively determined if the sample concentration is normalized, even with protein impurities. According to these characteristics, it could be possible to measure the interaction between purified hACE2 and S proteins in a solution mixed with a crude sample (e.g., serum addition). There are two important conditions for analyzing the interaction using FCS: the specific labeling of the fluorescent tag, and the expression of the full-length/intact form of the analyzed protein. We confirmed that the proteins in the samples existed in such a state using Western blotting before the FCCS measurements (Figure 2A). Spike proteins are composed of two subunits (S1 and S2). In this study, we used only the S1 subunit. CPM values obtained by FCCS analysis showed that mCherry-tagged S1 subunits exist as monomers (Figure 3C), suggesting that the S1 protein could be stable as monomers. Trimer formation of the spike protein has been reported [4,30]; however, the absence of the S2 subunit would not induce trimer formation. The monomer stability of the S1 subunit is considered advantageous for analyzing the interaction. The interaction between ACE2 and transmembrane-lacking S protein (S1 + S2 subunits; also called S ectodomain) of SARS-CoV-2 was weak compared to that between ACE2 and S1 subunit only, despite the trimer formation of S protein [5].