Engineering a cell-free biosensor signal amplification circuit with polymerase strand recycling

Area of Science:

• Synthetic biology
• Biotechnology
• Molecular biology

Background:

• Cell-free systems enable gene expression and sensing without living cells.
• Genetic circuits enhance cell-free systems for advanced functions.
• Need for improved signal amplification in cell-free biosensors.

Approach:

• Engineered a novel isothermal signal amplification circuit: polymerase strand recycling (PSR).
• Leveraged T7 RNA polymerase off-target transcription for nucleic acid input recycling in DNA strand displacement circuits.
• Developed design rules for PSR components and constructed modular biosensors.

Key Points:

• PSR enables direct sensing of RNA targets with nanomolar detection limits and high specificity.
• Applied PSR to allosteric transcription factor-based biosensors for small molecule detection.
• Demonstrated 3.6-4.6-fold enhancement in biosensor sensitivity (EC50) to sub-micromolar levels.

Conclusions:

• Polymerase strand recycling expands cell-free circuit capabilities for enhanced biosensing.
• The developed modular biosensors offer sensitive and specific detection of RNA and small molecules.
• PSR is anticipated to have broad applications in biotechnology and synthetic biology.