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Fluorescence light-up aptamers (FLAPs) are tools for RNA imaging, wherein the RNA of interest is appended with a FLAP sequence that can bind to a corresponding small-molecule fluorogen and enhance its fluorescence. The fluorescence properties of FLAPs have mostly been analyzed in bulk and described as the average of a large number of RNA–fluorogen complexes. In this study, we evaluated the feasibility of fluorescence correlation spectroscopy (FCS)- and fluorescence cross-correlation spectroscopy (FCCS)-based quantifications of FLAPs in a solution using Broccoli, a common FLAP, and its corresponding fluorogen, DFHBI-1T. We investigated the folding efficiency, photostability, and photophysical properties of the Broccoli–DFHBI-1T complex using their FCS/FCCS characteristics. With FCS, we observed that the fluorescence was affected by the affinity between Broccoli and DFHBI-1T and the folding (maturation) state of Broccoli RNA. Moreover, the FCCS measurement of ATTO647N-labeled Broccoli and its complex with DFHBI-1T revealed the proportion of the mature Broccoli–DFHBI-1T complex. The current FCS/FCCS-based study of Broccoli–DFHBI-1T provides a model for analyzing FLAPs and their fluorogen pairs at the single-molecule level.

Monitoring Molecular Properties of a Fluorescence Light-Up Aptamer Using Fluorescence Cross-Correlation Spectroscopy