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Related Experiment Video

Updated: May 17, 2025

Nanomanipulation of Single RNA Molecules by Optical Tweezers

Nanomanipulation of Single RNA Molecules by Optical Tweezers

Published on: August 20, 2014

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

Dian Chen¹, Jun Li², You Wu¹

¹State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.

Science Advances

|April 2, 2025

Summary

Researchers engineered a c-di-AMP riboswitch into a highly sensitive biosensor. This advancement enables improved detection of cyclic diadenosine monophosphate (c-di-AMP) and facilitates the study of STING immune pathway activators.

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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.