Kinetic-Gated Signal Transduction Decouples Binding Affinity in a Dual-Pocket Riboswitch RNA

Area of Science:

• Biochemistry
• Molecular Biology
• Biosensor Technology

Background:

• RNA's role extends beyond biology to biosensor design.
• Multi-pocket RNA sensors, like the tetrahydrofolate (THF) riboswitch, offer complex signal integration.
• The THF riboswitch binds different molecules but shows varied outputs, a puzzle in sensor mechanism.

Purpose of the Study:

• To elucidate the mechanism behind the decoupling of molecular recognition and sensor folding in the THF riboswitch.
• To understand how binding kinetics and ligand cooperativity influence RNA aptasensor performance.
• To provide insights for engineering advanced, logic-gated RNA aptasensors.

Main Methods:

• Developed a multi-modal analytical platform.
• Utilized engineered fluorescent sensors, RNA labeling, stopped-flow kinetics, microscale thermophoresis (MST), and single-molecule FRET.
• Investigated ligand binding kinetics and cooperativity.

Main Results:

• Functional activation of the THF riboswitch depends on target residence time (binding kinetics), not solely affinity.
• Concentration-dependent cooperativity among ligands acts as an allosteric amplifier.
• Broader dynamic range and lower detection thresholds were observed.

Conclusions:

• Binding kinetics are crucial for RNA aptasensor activation and function.
• Ligand cooperativity enhances sensor performance, enabling broader dynamic range and sensitivity.
• This research advances the engineering of sophisticated RNA aptasensors for multiplexed diagnostics.