Engineering and characterization of fluorogenic glycine riboswitches

Area of Science:

• Synthetic biology
• RNA nanotechnology
• Biophysics

Background:

• Riboswitches are genetic circuits that regulate gene expression in response to small molecules.
• Engineering novel riboswitches with tailored properties is crucial for biosensing applications.
• Fluorogenic riboswitches offer a direct readout of molecular binding events.

Purpose of the Study:

• To engineer a library of fluorogenic glycine riboswitches with diverse thermodynamic and kinetic properties.
• To optimize signal transduction between RNA sensor and actuator components.
• To establish a rapid screening method for generating functional riboswitches.

Main Methods:

• A three-part RNA approach was used, combining an RNA sensor (glycine aptamer), transmitter, and actuator (Spinach aptamer).
• A riboswitch library with variable transmitter sequences was screened using a microfluidic large-scale integration chip.
• Thermodynamic binding profiles, including glycine dissociation constants (Kd), were determined.
• Kinetic binding (kon) and dissociation (koff) rates were measured using the same microfluidic platform.

Main Results:

• Twelve fluorogenic glycine riboswitches with strong fluorescence responses were successfully engineered.
• Glycine dissociation constants (Kd) ranged from 99.7 to 570 μM.
• Kinetic rates (kon and koff) were determined to be in the order of 10^-3 s^-1 and 10^-2 s^-1, respectively.
• The engineered riboswitches exhibited a broad range of biophysical response properties.

Conclusions:

• Systematic screening of linked RNA parts using microfluidic chip technology is effective for rapid generation of fluorogenic metabolite riboswitches.
• This approach allows for the creation of riboswitches with tunable thermodynamic and kinetic characteristics.
• The engineered riboswitches have potential applications in metabolite detection and synthetic biology circuits.