Design of small molecule-responsive microRNAs based on structural requirements for Drosha processing.

Area of Science:

• Molecular Biology
• Synthetic Biology
• RNA Biology

Background:

• MicroRNAs (miRNAs) are crucial regulatory RNAs involved in gene silencing and cellular processes.
• Developing synthetic RNA-based systems is hindered by limited understanding of miRNA structure-function relationships.
• Existing systems lack fine-tuned control over miRNA-mediated gene silencing.

Purpose of the Study:

• To design novel ligand-responsive microRNAs (miRNAs) for controllable gene silencing.
• To establish a strategy for integrating sensing and regulatory functions into synthetic miRNAs.
• To enable titratable control over gene silencing based on user-defined inputs.

Main Methods:

• Leveraged the relationship between Drosha processing and miRNA basal segment structure.
• Integrated aptamers into the miRNA basal segments to create ligand-binding sites.
• Demonstrated inhibition of Drosha processing upon ligand binding to aptamers.
• Validated the strategy using three distinct aptamer-small molecule ligand pairs.

Main Results:

• Ligand binding to integrated aptamers successfully inhibited Drosha processing.
• This inhibition resulted in titratable control over gene silencing.
• The control strategy proved general across different aptamer-ligand pairs.
• The platform demonstrated potential for designing miRNA clusters and self-targeting miRNAs.

Conclusions:

• Developed a versatile platform for engineering ligand-responsive miRNAs.
• Enabled tunable gene silencing through user-defined small molecule inputs.
• The platform facilitates fine-tuning of regulatory strength and dynamics for synthetic gene circuits.
• Advances applications in biological research and medicine through precise RNA-based control.