MicroRNA-based single-gene circuits buffer protein synthesis rates against perturbations

Area of Science:

• Synthetic biology
• Molecular biology
• Biomedical engineering

Background:

• Mammalian gene expression control is challenging due to inherent noise and external perturbations.
• Existing methods struggle to achieve precise and stable transgene expression at the single-cell level.

Purpose of the Study:

• To demonstrate precise control of protein synthesis using a novel single-gene microRNA (miRNA)-based feed-forward loop (sgFFL).
• To investigate the buffering capacity of the sgFFL against transcriptional changes and noise.

Main Methods:

• Design and implementation of a minimal autoregulatory gene circuit (sgFFL) utilizing an intronic miRNA targeting its own transcript.
• Analysis of protein expression dynamics, adaptation, and steady-state levels in response to transcription rate changes.
• Quantification of transcriptional noise and cell-to-cell variability reduction by the sgFFL.

Main Results:

• The sgFFL generated a transient protein pulse and adapted to a lower steady state, independent of stimulus size.
• Protein levels were effectively buffered against changes in transcription rate.
• The sgFFL significantly reduced protein synthesis noise, with up to 5-fold lower variability compared to unregulated controls.
• Noise buffering correlated with miRNA-target interaction strength.

Conclusions:

• The single-gene feed-forward loop (sgFFL) provides a robust and generalizable mechanism for precise gene expression control.
• This motif offers a powerful platform for engineering stable synthetic gene circuits and understanding natural regulatory networks.
• The sgFFL architecture effectively mitigates noise and perturbations, enhancing the reliability of protein production.