# Monitoring Molecular Properties of a Fluorescence Light-Up
## 1 Introduction  
RNA plays an important role in gene expression and regulation. Studying RNA-mediated biological systems increases the understanding of life processes and helps in disease diagnosis and therapy. Thus, visualization approaches must be developed to reveal cell RNA localization and detect disease-related RNA in solution.  
Fluorescence light-up aptamers (FLAPs) are among the most promising candidates for RNA visualization. FLAP is an RNA aptamer that can specifically bind to a corresponding small-molecule fluorogen. Such fluorogens are usually cell-permeable and essentially non-fluorescent. However, they become highly fluorescent upon binding to the partner FLAP. Thus, FLAP technology provides a simple and promising approach for live-cell RNA imaging by fusing a FLAP sequence to the RNA of interest. For example, Broccoli, a 49-base RNA aptamer, is a FLAP that binds to DFHBI-1T and enhances its green fluorescence [1; 2; 3]. Broccoli-DFHBI-1T was developed as an improved version of Spinach-DFHBI, and is used in many intracellular and extracellular applications because of its compactness and high fluorescence in physiological environments. In many cases, the fluorescence properties of FLAPs, including Broccoli, have been analyzed in bulk and described as an average of a large number of FLAP-fluorogen complexes, except for a few methods, such as super-resolution imaging. Thus, the fluorescence properties of a FLAP complex with a fluorogen have not been analyzed at the molecular level.  
Fluorescence correlation spectroscopy (FCS) is a technique that reveals molecular diffusion dynamics and quantifies the number of molecules of fluorescently labeled target molecules at the single-molecule level [4; 5; 6]. FCS is mainly applied for the investigationof particle size change caused by molecular interaction, enzymatic activity, and aggregation, and for the quantification of fluorescently labeled biomolecules, including RNA, in solutions and living cells [7; 8; 9; 10]. In addition, FCS can measure the brightness of a single molecule or particle. In the case of bulk analysis, the obtained fluorescence intensity is a convolution of molecular concentration and single-molecule brightness. Using FCS, we can obtain information about the molecular brightness and concentration of fluorescent molecules individually.  
Fluorescence cross-correlation spectroscopy (FCCS) is a dual-color extension of FCS [11; 12]. FCCS can monitor the interaction between two fluorescently labeled molecules directly by observing the coincidence of fluctuations between two fluorescence intensity profiles. The strength of the molecular interaction is described as the dissociation constant (\(\rm K_{d}\)), which is calculated from the concentrations of each target molecule and complex. In FCCS, concentrations of two kinds of target molecule from autocorrelation curves and the concentration of binding complexes from the cross-correlation curve can be used to calculate the \(\rm K_{d}\) value [13; 14]. [PERSON] et al. applied the FCCS technique to DNA degradation studies in living cells [15]. FCCS can monitor molecular binding directly in solutions and in living cells.  
This report evaluates the feasibility of FCS/FCCS measurements of FLAP-fluorogen pairs, using Broccoli-DFHBI-1T as a model in the conventional FCS/FCCS measurement conditions. In addition, we investigated the folding efficiency, photostability, and photophysical properties of Broccoli-DFHBI-1T by taking advantage of FCS/FCCS, which have not been analyzed in detail at the single-molecule level.