# Monitoring Molecular Properties of a Fluorescence Light-Up
## 3 Results and Discussion
Red line, 1 \(\upmu\)M Broccoli; blue line, 2 \(\upmu\)M Broccoli; green line, 5 \(\upmu\)M Broccoli; black line, no RNA control measurement (only in **a**). Error bars show standard deviation in fitting analysis (derived via Bootstrap method). Next, we compared the brightness and size of Broccoli-DFHBI-1T to conventional fluorescent molecules. Fluorescein (pH 9.5) and purified 6 xhis-AcGFP solutions were prepared and measured under the same conditions as the Broccoli-DFHBI-1T measurement (Figure 4a). The obtained diffusion time of Broccoli-DFHBI-1T was slightly smaller than that for 6 xhis-AcGFP (Figure 4b). This result indicated that the size of Broccoli-DFHBI-1T was approximately 3 nm. The obtained CPM values show that the brightness of AcGFP was four times that of Broccoli-DFHBI-1T under our measurement conditions (excitation: 488 nm, emission: 500-750 nm) (Figure 3c). However, the result is not consistent with a previous report, where the reported quantum yield (Q\({}_{y}\) = 0.41) of Broccoli-DFHBI-1T [20] was half of that of AcGFP (Q\({}_{y}\) = 0.82), and the molar extinction coefficient (\(\epsilon\) = 28,900) of Broccoli-DFHBI-1T [20] was similar to that of AcGFP (\(\epsilon\) = 32,500). This may be due to the excitation efficiency at 488 nm. Furthermore, the folding state of Broccoli fluctuates dynamically, and the fluorescence emission rate of a single molecule is unstable, depending on the transient folding state. If the Broccoli RNA molecule and Broccoli-DFHBI-1T complex does not form an appropriate structure, the complex cannot brightly fluoresce, which affects the single-molecule measurement of the FCS. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline  & **RCA** & **RCA** & **C\({}_{\text{ppm}}\)** & **C\({}_{\text{rd}}\)** & **C\({}_{\text{complex}}\)** & **Apparent K\({}_{\text{d}}\)** & **Estimated C\({}_{\text{complex}}\)** & **Folding Efficiency** \\  & **Ge(0)/Gr(0)** & **Ge(0)/G(0)** & **(nM)** & **(nM)** & **(nM)** & **(nM)** & **(nM)** & **K\({}_{\text{d}}\)** = 360 nM (nM)** & **(C\({}_{\text{complex}}\)/Estimated C\({}_{\text{complex}}\))** \\ \hline Broccoli- & & & & & & & & & \\ ATTO647N & 0.987 & 0.198 & 75.3 & 260.5 & 60.6 & & & & \\ (1 uM) & \(\pm\) 0.041 & \(\pm\) 0.011 & \(\pm\) 0.0 & \(\pm\) 5.1 & \(\pm\) 2.5 & 42,961 * & 251.3 & 0.241 \\ (10 uM) & & & & & & & & \\ Bruccoli- & & & & & & & & \\ ATTO647N & 0.923 & 0.137 & 50.2 & 245.0 & 37.9 & & & & \\ (1 uM) & \(\pm\) 0.053 & \(\pm\) 0.009 & \(\pm\) 2.7 & \(\pm\) 8.3 & \(\pm\) 3.6 & 6472 * & 170.8 & 0.222 \\ (1 uM) & & & & & & & & \\ \hline \end{tabular}
\end{table} 
Table 1: Relative cross-correlation amplitude (RCA), measured concentrations, apparent K\({}_{\text{d}}\) in the FCCS measurement, and estimated folding efficiency of Broccoli molecules. * Values when Broccoli’s folding efficiency is not considered. Figure 3: Direct measurement of the interaction between ATTO647N-labeled Broccoli RNA and DFHBI-1T using fluorescence cross-correlation spectroscopy (FCCS). Autocorrelation curves of green channel (Broccoli–DFHBI-1T fluorescence, excitation: 488 nm, 80 uW) and red channel (ATTO647N fluorescence, excitation: 633 nm, 2 uW), and cross-correlation curve between two channels with the 10 uM (**a**) and 1 uM (**b**) concentration of DFHBI-1T. (**c**) Diagram of possible states of ATTO647N-labeled Broccoli RNA and DFHBI-1T in the measurement system.