# Monitoring Molecular Properties of a Fluorescence Light-Up
## 3 Results and Discussion  
To determine whether FLAPs can be applied to fluorescence correlation spectroscopy measurements, we performed the FCS measurements of Broccoli-DFHBI-1T as a model with various laser powers. Clear autocorrelation curves were obtained at a relatively high laser power (Figure 1a), and the model equation for one-component diffusion fitted the curves well. At a moderate laser power, the fluorescence signal decreased within 60 s, after which the signal plateaued at two-thirds of the initial value and was not completely quenched (Figure 1b), thus indicating that the decrease in fluorescence was due to a reversible reaction rather than irreversible photobleaching. DFHBI, an analog of DFHBI-1T, undergoes _cis_-to-_trans_ photoisomerization upon excitation and attenuates its fluorescence [18; 19]. Since _trans_-DFHBI reverts to _cis_-DFHBI because of thermal relaxation, the fluorescence reaches a plateau at equilibrium during reversible photoisomerization. Therefore, we hypothesized that reversible photoisomerization occurs in Broccoli-DFHBI-1T. To verify this hypothesis, a second measurement was carried out with the same sample, 5 min after first measurement. The signal completely recovered to the initial value of the first measurement, and a similar decay was then observed. The results are consistent with previous reports, and indicate that DFHBI-1T is involved in reversible _cis-trans_ photoisomerization. Moreover, Broccoli-DFHBI-1T had sufficient brightness to be applied to FCS to carry out single-molecule-level analysis. Next, we monitored the number of fluorescent molecules detected by forming a complex of Broccoli and DFHBI-1T. The FCS measurements were carried out using a mixture of Broccoli-DFHBI-1T at different concentrations (Figure 2). Figure 2a shows the average photon count rate of the FCS measurements. The count rate increased depending on the DFHBI-1T concentration and the Broccoli concentration in the samples. The molar concentration of the fluorescent Broccoli-DFHBI-1T complex was then calculated from the molecular number detected in the effective confocal volume (Figure 2b). The effective confocal volume was estimated to be 0.40 fL via the diffusion measurement of the ATTO488 reference solution. FCS can monitor single-molecule brightness as counts per molecule (CPM) (Figure 2c). The CPM showed a plateau value when the concentration of DFHBI-1T was markedly higher than that of Broccoli. This plateau CPM value indicates that the FCS measurements in this concentration range are stable, and that the determined concentration values are reliable. The concentration of the fluorescent Broccoli-DFHBI-1T complex estimated by FCS was 10% to 20% of the prepared Broccoli concentration. Since the K\({}_{\text{d}}\) value of Broccoli-DFHBI-1T was proposed to be 360 nM [1], the observed difference in concentration cannot be explained only by the degree of Broccoli-DFHBI-1T binding. This difference can be attributed to imperfect folding and the fluctuation of the folding state of Broccoli RNA molecules. Although the results of FCS measurements (Figure 2) indicated that the actual fraction of the Broccoli-DFHBI-1T complex is less than that estimated from its K\({}_{\text{d}}\) value and the concentrations of each component, there was no direct evidence. Thus, we attempted FCCS measurements to directly monitor and quantify the molecular interactions between Broccoli and DFHBI-1T. ATTO647N-labeled Broccoli RNA (1 uM) was chemically synthesized and measured with DFHBI-1T (1 uM or 10 uM) using FCCS. The autocorrelation curves in the green and red channels and cross-correlation curves between them were successfully obtained (Figure 3a,b). The concentrations of total RNA (red channel, C\({}_{\text{red}}\)), fluorescent Broccoli-DFHBI-1T (green channel, C\({}_{\text{green}}\)), and the fraction of the complex that fluoresced green and red (cross-correlation, C\({}_{\text{complex}}\)) were determined.