# Monitoring Molecular Properties of a Fluorescence Light-Up
## 3 Results and Discussion
Table 1 shows the C\({}_{\text{complex}}\)  
Figure 1: Fluorescence correlation spectroscopy (FCS) measurements of Broccoli–DFHBI-1T (1 uM each). (**a**) Autocorrelation curves of Broccoli–DFHBI-1T with various laser powers. The autocorrelation curves were obtained after the fluorescence signal plateaued. (**b**) Fluorescence intensity profiles during two sequential FCS measurements (60 s, thrice each) of the same sample. Excitation laser power was 15 uW. \(\text{C}_{\text{green}}\), \(\text{C}_{\text{red}}\), \(\text{K}_{\text{d}}\), and the folding efficiency, derived from the RCA values with the standard deviation (\(n=3\)). In the case of Broccoli-DFHBI-1T, DFHBI-1T fluoresced only when making a complex with Broccoli; thus, \(\text{C}_{\text{green}}\) and \(\text{C}_{\text{complex}}\) show similar values. This result indicates that the labeling efficiency of ATTO647N-Broccoli was approximately 100%. The estimated concentrations of RNA from the FCS/FCCS were lower than the prepared concentrations. This is possibly due to the difference in the quantification method and adsorption to the tubes and glass surface. Using actual FCCS concentrations and the prepared DFHBI-1T concentration (1 \(\upmu\)M), the apparent \(\text{K}_{\text{d}}\) between Broccoli and DFHBI-1T was calculated as 6472 nM. However, this is different from the reported \(\text{K}_{\text{d}}\) value (360 nM) [1]. We consider that this difference in \(\text{K}_{\text{d}}\) values is due to the folding efficiency of Broccoli. Since the total RNA was used for the calculation, the presence of misfolded RNAs that cannot induce DFHBI-1T fluorescence can directly affect the obtained \(\text{K}_{\text{d}}\) value (Figure 3c). Assuming that the Kd value is 360 nM, as reported previously, the estimated \(\text{C}_{\text{complex}}\) in our measurement condition can be expected to be 170 nM. By comparing it with the measured \(\text{C}_{\text{complex}}\) (37.9 nM) in our FCCS measurement, we calculated the folding efficiency of Broccoli RNAs as 22.2%. A similar folding efficiency (24.1%) was obtained when DFHBI-1T was 10 \(\upmu\)M. [PERSON] et al. [1] reported that the folding efficiency of Broccoli without the tRNA scaffold was 40-50%; our measured value was lower, probably due to differences in the pre- and post-Broccoli sequences. The gap between the pre-measured and actual concentrations of Broccoli RNA in samples would be another reason. [PERSON] et al. calculated the folding efficiency of Broccoli using the pre-measured concentration. However, the actual RNA concentration in a sample should be vary from the pre-measured one because RNA is easily adsorbed on tubes and chips, as we observed in this study (Table 1, \(\text{C}_{\text{red}}\)). Thus, we directly measured the total concentration of Broccoli RNA in a sample using ATTO647N dye fused to the RNA. As the folding efficiency is calculated based on the total RNA concentration, the difference in the concentration measurement can directly lead to the different values. We believe that our study provides valuable information in the structural study of Broccoli RNA in solution. This study directly measured total Broccoli RNA labeled with ATTO647N and Broccoli-DFHBI-1T complexes that fluoresced simultaneously. In bulk analysis, the actual concentration of target molecules in the system is difficult to determine. In contrast, the absolute molecular number can be obtained, and the value can be converted into concentrations in FCS/FCCS. This is the advantage of the FCS/FCCS technique for investigating concentration-dependent phenomena, such as molecular interactions. Therefore, it was used to evaluate the interaction of a typical FLAP (Broccoli-DFHBI-1T) and understand its folding efficiency. Figure 2: Fitting analysis of Broccoli–DFHBI-1T for different concentrations of Broccoli and DFHBI-1T. (**a**) Count rate. (**b**) FCS concentration calculated from the number particles in the measurement volume. (**c**) Counts per molecule (CPM).