Engineering Riboswitches in Vivo Using Dual Genetic Selection and Fluorescence-Activated Cell Sorting

Area of Science:

• Synthetic Biology
• Molecular Biology
• RNA Biology

Background:

• Riboswitches are noncoding RNAs that regulate gene expression by binding small molecule effectors.
• Existing riboswitches are limited, hindering their application in synthetic biology.
• Novel riboswitches are needed to expand the toolkit for precise gene control.

Purpose of the Study:

• To develop a novel platform for discovering new riboswitches.
• To identify and characterize riboswitches that respond to specific small molecules.
• To demonstrate the utility of directed evolution for improving riboswitch function in vivo.

Main Methods:

• A riboswitch discovery platform was created using dual genetic selection and fluorescence-activated cell sorting.
• A large library (10^8 sequences) with a modified aptamer domain was screened for riboswitch activity.
• Directed evolution, including random mutagenesis, was employed to enhance riboswitch performance.

Main Results:

• Novel theophylline-responsive riboswitches were identified from the engineered library.
• The best identified riboswitch (Hit 3-5) showed 2.3-fold activation of gene expression.
• Further optimization through directed evolution yielded riboswitches with nearly 3-fold activation.

Conclusions:

• The developed platform is effective for discovering novel riboswitches.
• Directed evolution can successfully improve the in vivo function of engineered riboswitches.
• This work represents the first in vivo directed evolution of an aptamer domain for functional riboswitch generation.