Structural dynamics-guided engineering of a riboswitch RNA for evolving c-di-AMP synthases

Area of Science:

• Molecular Biology
• Biochemistry
• Immunology

Background:

• Cyclic diadenosine monophosphate (c-di-AMP) is a crucial second messenger activating the stimulator of interferon genes (STING) pathway.
• Characterizing c-di-AMP synthases is challenging due to the absence of effective detection tools.
• Natural c-di-AMP riboswitches, while promising RNA biosensors, have limitations including poorly understood dynamics and an "OFF" output.

Purpose of the Study:

• To engineer a novel c-di-AMP biosensor with an "ON" genetic output.
• To enhance the sensitivity and utility of c-di-AMP detection.
• To develop a platform for high-throughput evolution of c-di-AMP synthases.

Main Methods:

• Single-molecule fluorescence labeling and analysis to probe riboswitch conformational changes.
• Integration of fluorescence titration, mutagenesis, and in vivo assays.
• Development of a strand displacement strategy for biosensor engineering.

Main Results:

• Engineered a c-di-AMP riboswitch into a functional "ON" output biosensor.
• Achieved a 50-fold improvement in the detection limit for c-di-AMP.
• Demonstrated the biosensor's utility in high-throughput in vivo evolution of c-di-AMP synthases.

Conclusions:

• The engineered biosensor overcomes limitations of natural riboswitches for c-di-AMP detection.
• This tool significantly advances the study of c-di-AMP metabolism and STING pathway regulation.
• The developed platform enables efficient discovery and optimization of c-di-AMP synthases.