Bright and pH-sensitive Baby Spinach aptamer with RNA triplex fusion

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Intercellular pH is a critical factor in cellular processes.
• Existing pH probes often lack sensitivity or require complex modifications.
• RNA aptamers offer a versatile platform for developing molecular probes.

Purpose of the Study:

• To develop a novel fluorescent RNA aptamer for sensitive monitoring of intercellular pH.
• To engineer a pH-sensitive nanoswitch using an RNA triplex structure.
• To enhance the fluorescence properties of an existing aptamer for improved pH sensing.

Main Methods:

• Design and synthesis of a triplex-fused fluorescent RNA aptamer (Bright Baby Spinach aptamer).
• Characterization of pH-sensitive structural changes in the RNA triplex.
• In-cell experiments to evaluate the probe's response to intercellular pH variations.
• Spectroscopic analysis to quantify fluorescence enhancement.

Main Results:

• The developed RNA triplex structure functions as a pH-nanoswitch, exhibiting pH-dependent structural changes.
• The Bright Baby Spinach aptamer demonstrated rapid and sensitive responses to intercellular pH fluctuations.
• A significant fluorescence enhancement (up to 13-fold) was observed compared to the original aptamer.
• The RNA triplex structure likely promotes optimal folding for ligand binding and fluorescence.

Conclusions:

• The Bright Baby Spinach aptamer is a highly fluorescent and pH-sensitive probe.
• This aptamer enables rapid and sensitive detection of intracellular pH changes.
• The engineered aptamer holds potential for advanced cellular pH monitoring applications.