Repurposing an adenine riboswitch into a fluorogenic imaging and sensing tag

Area of Science:

• Molecular Biology
• Biochemistry
• Synthetic Biology

Background:

• Fluorogenic RNA aptamers are crucial for creating fluorescent RNA and metabolite sensors.
• Naturally occurring aptamers fold well and change conformation with metabolites, but engineered fluorogenic aptamers often have poor folding, limiting cellular fluorescence.

Purpose of the Study:

• To engineer a well-folded, cell-compatible fluorogenic aptamer by evolving a natural adenine riboswitch.
• To develop a novel RNA-based sensor for precise metabolite quantification within cells.

Main Methods:

• Generated a large library (~10^15) of adenine aptamer-like RNAs with randomized adenine-binding pockets.
• Selected for aptamers that bind metabolites and activate fluorescence, leading to the discovery of Squash.
• Fused the Squash aptamer to a S-adenosylmethionine (SAM)-binding aptamer for metabolite sensing.

Main Results:

• Squash demonstrates significantly improved in-cell folding and metabolite-dependent fluorescence activation.
• A Squash-based ratiometric sensor enabled quantitative SAM measurements in cells.
• The sensor revealed cell-to-cell variability in SAM levels and identified metabolic origins of SAM.

Conclusions:

• Naturally occurring aptamer folding can be leveraged to engineer robust, cell-compatible fluorogenic aptamers.
• The Squash aptamer represents a significant advancement for RNA-based biosensors and fluorescent RNA applications.
• This work facilitates deeper understanding of cellular metabolism and enables new tools for molecular biology.