Artificial dynamic structure ensemble-guided rational design of a universal RNA aptamer-based sensing tag

Area of Science:

• Synthetic Biology
• RNA Biology
• Biotechnology

Background:

• Artificially functional RNAs, like fluorogenic RNA aptamer (FRApt)-based biosensing tags, offer potential in biological applications.
• Current limitations include a lack of understanding of dynamic structure ensembles and universal design principles.

Purpose of the Study:

• To develop a universal fluorogenic RNA biosensing tag with a modular design.
• To enable low-background, highly selective imaging of biomolecules in living cells.
• To create an effective tool for screening single guide RNA (sgRNA) in CRISPR-mediated gene editing.

Main Methods:

• Development of an artificial RNA structure ensemble and an RNA reconstitution model named "SSPepper-Apt."
• Utilizing various target-recognizing RNA motifs for modular tag generation.
• Employing single guide RNA (sgRNA) as a recognition motif for CRISPR applications.

Main Results:

• SSPepper-Apt successfully generated universal fluorogenic RNA sensing tags.
• The tags enabled modular construction for selective imaging of metabolites, peptides, and proteins in living cells.
• SSPepper-Apt demonstrated utility as an sgRNA screening tool for gene editing by detecting Cas9-sgRNA complex assembly.

Conclusions:

• The developed fluorogenic RNA-sensing tag offers a universal approach for constructing functional RNA systems.
• This method circumvents laborious sequence combination processes.
• It expands the utility of synthetic biological tools in biological research and applications.