Abstract
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.
Highlights
RNA plays an important role in gene expression and regulation
To determine whether Fluorescence light-up aptamers (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
The results are consistent with previous reports, and indicate that DFHBI-1T is involved in reversible cis–trans photoisomerization
Summary
RNA plays an important role in gene expression and regulation. Studying RNAmediated biological systems increases the understanding of life processes and helps in disease diagnosis and therapy. 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 bind to a corresponding small-molecule fluorogen. Such fluorogens are usually cell-permeable and essentially non-fluorescent. They become highly fluorescent upon binding to the partner FLAP. Broccoli, a 49-base RNA aptamer, is a FLAP that binds to DFHBI-1T and enhances its green fluorescence [1,2,3]. The fluorescence properties of a FLAP complex with a fluorogen have not been analyzed at the molecular level
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