Engineering a Synthetic Dopamine-Responsive Riboswitch for In Vitro Biosensing

Area of Science:

• Biotechnology
• Molecular Biology
• Chemical Sensing

Background:

• Natural allosteric transcription factors enable point-of-use molecular sensing with cell-free gene expression (CFE).
• Current methods are limited by the availability of characterized protein-based sensors, restricting the range of detectable analytes.
• Developing RNA aptamer-based sensors offers an alternative but has seen limited success in portable CFE systems.

Purpose of the Study:

• To engineer functional dopamine-sensing riboswitches from dopamine-binding aptamers for CFE systems.
• To establish a method for converting *in vitro*-generated RNA aptamers into fieldable molecular diagnostics.
• To enable the detection and semi-quantification of dopamine in human urine.

Main Methods:

• Conversion of dopamine-binding aptamers into riboswitches that regulate gene expression.
• Integration of engineered riboswitches into a freeze-dried CFE system.
• Development of a diagnostic assay for dopamine detection in human urine.

Main Results:

• Successfully converted dopamine-binding aptamers into functional riboswitches for gene expression control.
• Demonstrated ligand-inducible gene expression regulation in a freeze-dried CFE system.
• Developed a novel assay for direct detection and semi-quantification of dopamine in human urine samples.

Conclusions:

• The study presents a broadly applicable strategy for converting *in vitro*-generated RNA aptamers into fieldable molecular sensors.
• This work advances the development of portable cell-free diagnostics for various analytes.
• The engineered riboswitches offer a promising platform for point-of-use chemical sensing applications.