Genetically encoded RNA-based sensors with Pepper fluorogenic aptamer.

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Intracellular molecule sensing is vital for understanding physiological functions.
• Conventional fluorescent protein sensors exist, but RNA-based sensors offer unique advantages.
• Existing RNA sensors often contain RNA G-quadruplex (RG4) motifs, potentially limiting their efficacy in vivo.

Purpose of the Study:

• To develop novel RNA-based sensors utilizing Pepper, a fluorogenic RNA lacking RG4 motifs.
• To engineer and validate sensors for diverse intracellular targets including metabolites, proteins, and metal ions.
• To demonstrate the application of these sensors in monitoring cellular pathways and evaluating drug efficacy in living cells.

Main Methods:

• Engineering RNA-based sensors using the Pepper fluorogenic RNA platform.
• In vitro and in vivo validation of sensor performance, including activation and selectivity.
• Development of a ratiometric sensor for S-adenosylmethionine (SAM) to assess drug inhibition.

Main Results:

• Successfully developed Pepper-based RNA sensors for various targets, functioning both in vitro and in living cells.
• Demonstrated high activation, selectivity, universality, and robustness of the engineered sensors.
• Successfully monitored S-adenosylmethionine (SAM) biosynthesis, methionine adenosyltransferase (MATase) activity, and gene expression spatiotemporally.
• Quantified the inhibition efficacy of a MATase inhibitor using a ratiometric SAM sensor.

Conclusions:

• Pepper-based RNA sensors provide a versatile and robust platform for imaging diverse cellular targets.
• These sensors enable real-time monitoring of molecular signaling pathways and drug responses in living cells.
• The developed sensors significantly advance the capabilities for intracellular molecule detection and pathway analysis.