A Novel Aptamer-Based Fluorescent Biosensor for Imaging SIRT2 in Live Cells and Screening Its Modulators

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Sirtuin 2 (SIRT2) is implicated in various diseases, including diabetes and neurodegenerative disorders, making it a significant therapeutic target.
• Current methods lack the ability to image SIRT2 protein in live cells, hindering rapid detection and modulator screening.

Purpose of the Study:

• To develop a novel "turn-on" fluorescent biosensor for effective imaging of SIRT2 in live cells.
• To establish a platform for high-throughput screening of SIRT2 modulators using the developed biosensor.

Main Methods:

• Systemic evolution of ligands by exponential enrichment (SELEX) was employed to discover high-affinity aptamers for SIRT2.
• A "turn-on" fluorescent biosensor was fabricated using a selected aptamer (Apt 45) conjugated with fluorescent dyes and gold nanospheres.
• The biosensor's performance was evaluated for specificity, cytotoxicity, and imaging capabilities in different cell lines.

Main Results:

• Twelve aptamers with high affinity (Kd = 123.3-154.5 nM) to SIRT2 were identified, with Apt 45 selected for biosensor fabrication.
• The aptamer-based biosensor demonstrated excellent specificity and low cytotoxicity, successfully imaging SIRT2 in Schwann, H9c2, and HUVECs cells.
• The biosensor platform facilitated the identification of three SIRT2 modulators (astragaloside II, chlorogenic acid, tanshinone IIA) that enhanced SIRT2 levels in high glucose/lipid-damaged Schwann cells.

Conclusions:

• An aptamer-based "turn-on" fluorescent biosensor was successfully developed for live-cell imaging of SIRT2.
• This biosensor provides a valuable tool for high-throughput screening of protein modulators at the cellular level.
• The developed approach offers a universal strategy for screening modulators of various target proteins by aptamer replacement.