Double-stemmed and split structural variants of fluorescent RNA Mango aptamers

Area of Science:

• Molecular Biology
• Biochemistry
• Biotechnology

Background:

• Fluorogenic aptamers, like RNA Mango, are valuable for RNA quantification and tracking.
• The single-stem structure of existing RNA Mango aptamers limits design modifications.
• G-quadruplex structures are key to RNA Mango's function.

Purpose of the Study:

• To engineer novel RNA Mango aptamer variants with enhanced structural features.
• To improve fluorescence properties and expand design possibilities for RNA Mango aptamers.
• To explore applications in RNA imaging and detection of RNA-RNA interactions.

Main Methods:

• Designed and synthesized double-stemmed RNA Mango aptamer variants.
• Performed fluorescence saturation analysis to quantify binding and brightness.
• Introduced and analyzed mutations in the second stem's linker region.
• Demonstrated the function of a split, bimolecular Mango aptamer.

Main Results:

• Double-stemmed RNA Mango variants exhibited up to ~75% brighter fluorescence compared to the original.
• Mutations in the second linker suggested indirect ligand interaction influencing fluorescence.
• The second stem's linker offers potential for rational design and reselection.
• A bimolecular Mango aptamer was successfully created and functional in vitro.

Conclusions:

• New double-stemmed RNA Mango aptamers offer enhanced fluorescence and greater designability.
• The second stem provides a platform for engineering improved aptamer performance.
• Bimolecular Mango aptamers show promise for detecting RNA-RNA interactions.
• These advancements expand the utility of RNA Mango aptamers for diverse biological applications.