Rational Modular RNA Engineering Based on In Vivo Profiling of Structural Accessibility.

Area of Science:

• Molecular Biology
• Synthetic Biology
• RNA Biology

Background:

• Bacterial small RNAs (sRNAs) are key regulators of gene expression.
• Current engineered sRNAs primarily target synthetic genes, limiting applications in native networks.

Purpose of the Study:

• To develop a rational, modular RNA engineering strategy for tuning the regulatory activity of multisubstrate sRNAs.
• To enable differential control of native gene networks using engineered sRNAs.

Main Methods:

• Utilized in vivo structural accessibility data to guide engineering of the CsrB sRNA.
• Modularly recombined selected substructures to create a library of CsrB variants.
• Employed fluorescent translational reporter assays to measure regulatory activity.

Main Results:

• Generated 21 CsrB variants achieving a 5-fold gradient of control over native Csr network targets.
• Identified less conserved local structures in multisubstrate sRNAs as promising targets for rational engineering.
• Demonstrated the potential for tuning global sRNA regulators for metabolic engineering.

Conclusions:

• A modular RNA engineering approach, informed by in vivo structural data, can precisely tune sRNA function for native gene networks.
• This method offers a powerful tool for engineering global sRNA regulators in diverse biological applications, including metabolic engineering.