Scaffolding Light-Up Aptamers on DNA Nanostructures for Fluorescence Enhancement.

RNA-based fluorescent light-up aptamers (FLAPs) have been progressively developed as imaging probes because of their high signal-to-noise ratio. However, it remains a challenge to use these light-up aptamers due to their poor folding and stability. Leveraging DNA nanotechnology, we investigated whether a DNA origami template could improve folding and further enhance the functionality of FLAPs, namely, the corresponding fluorescence intensities. We utilized aptamer Broccoli and its cognate fluorogen DFHBI-1T as a model. When singular aptamer Broccoli was scaffolded on DNA origami, DNA brick-based nanostructures, DNA double helices, and even on structures as simple as a DNA hairpin stem, our results showed that the fluorescence intensities could be significantly enhanced. These findings show a positive correlation between the fluorogen activity of light-up aptamers and the DNA stem length, potentially mediated by the improved structural stability of the DNA stem, as determined by their simulated thermodynamic properties. Our studies provide a new method to design and enhance the fluorescence behavior of FLAPs, especially structures with a G-quadruplex-based fluorogen recognition region.