A Simple Molecular Rotor for Defining Nucleoside Environment within a DNA Aptamer-Protein Complex

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysical Chemistry

Background:

• Aptamers, like the thrombin binding aptamer (TBA), are crucial in molecular recognition.
• Understanding aptamer-protein interactions at the nucleoside level is key for aptamer-based diagnostics and therapeutics.
• The thrombin binding aptamer (TBA) forms a G-quadruplex (GQ) structure upon folding and protein binding.

Purpose of the Study:

• To investigate the utility of 5-furyl-2 ahydrodeoxyuridine ((Fur)dU) as a dual-mode fluorescent probe.
• To map the microenvironment of thymidine (T) residues within the thrombin binding aptamer (TBA) during G-quadruplex (GQ) folding and thrombin binding.
• To leverage the molecular rotor and emissive properties of (Fur)dU for sensing aptamer-protein interactions.

Main Methods:

• Incorporation of the 5-furyl-2 ahydrodeoxyuridine ((Fur)dU) nucleobase into the thrombin binding aptamer (TBA).
• Utilizing the emissive sensitivity of (Fur)dU to microenvironment polarity changes.
• Employing the molecular rotor characteristics of (Fur)dU for fluorescence sensing of solvent rigidity changes.
• Analyzing fluorescence changes to distinguish T bases in different structural contexts (solvent-exposed in GQ vs. apolar duplex) and to identify specific protein-binding sites.

Main Results:

• The (Fur)dU probe successfully distinguished between solvent-exposed T bases within the G-quadruplex (GQ) structure and those in an apolar duplex environment.
• The molecular rotor property of (Fur)dU enabled a turn-on fluorescence switch, specifically identifying T residues involved in thrombin binding.
• The study demonstrated the ability of (Fur)dU to map the microenvironment of T loop residues in the TBA during folding and protein interaction.

Conclusions:

• 5-furyl-2 ahydrodeoxyuridine ((Fur)dU) is a versatile fluorescent probe for characterizing aptamer microenvironments.
• The dual probing capabilities of (Fur)dU (polarity sensitivity and molecular rotor character) are effective for mapping aptamer-protein interactions.
• This nucleoside probe offers a valuable tool for developing modified aptamers for diagnostic and therapeutic applications.