Scaffolding Light-Up Aptamers on DNA Nanostructures for Fluorescence Enhancement

Area of Science:

• Biotechnology
• Molecular Biology
• Nanotechnology

Background:

• RNA-based fluorescent light-up aptamers (FLAPs) offer high signal-to-noise ratios for imaging.
• Challenges exist in FLAP folding and stability, limiting their application.
• DNA nanotechnology presents a potential solution for enhancing FLAP performance.

Purpose of the Study:

• To investigate if DNA origami templates can improve the folding and enhance the fluorescence intensity of FLAPs.
• To explore the relationship between DNA nanostructure scaffolding and FLAP functionality.
• To optimize FLAP design for improved imaging probe applications.

Main Methods:

• Utilized aptamer Broccoli and its cognate fluorogen DFHBI-1T as a model system.
• Scaffolded aptamer Broccoli onto various DNA nanostructures, including DNA origami, DNA bricks, DNA double helices, and DNA hairpin stems.
• Analyzed fluorescence intensities and simulated thermodynamic properties to assess structural stability.

Main Results:

• Scaffolding aptamer Broccoli on DNA nanostructures, even simple DNA hairpin stems, significantly enhanced fluorescence intensities.
• A positive correlation was observed between DNA stem length and fluorogen activity of the light-up aptamers.
• Improved structural stability of the DNA stem, supported by thermodynamic simulations, likely mediates the enhanced fluorescence.

Conclusions:

• DNA origami and related DNA nanostructures can effectively enhance the folding and fluorescence of RNA-based light-up aptamers.
• This approach offers a novel strategy for designing and improving FLAPs, particularly those with G-quadruplex-based fluorogen recognition regions.
• The findings provide a new method to boost the performance of FLAPs as advanced imaging probes.