Visualizing NEK9 in action: aptamer-based fluorescent probes for real-time live-cell imaging

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Live-cell imaging is crucial for understanding protein dynamics in biological research and medicine.
• Conventional labeling techniques face challenges like poor probe permeability and cytotoxicity, hindering effective live-cell imaging.

Purpose of the Study:

• To develop and validate a novel aptamer-based fluorescent probe for real-time imaging of NEK9 kinase in living cells.
• To overcome the limitations of existing live-cell imaging methods.

Main Methods:

• Identified a DNA aptamer (Apt-011) that selectively binds NEK9 using in vitro capture-SELEX.
• Engineered a 'signal-on' aptamer-based fluorescent probe utilizing aptamer-induced conformational changes to separate fluorophore-quencher pairs.
• Validated the probe's performance in live-cell imaging, assessing NEK9 visualization and cytotoxicity.

Main Results:

• Successfully identified Apt-011, a DNA aptamer with high specificity for NEK9.
• Developed a novel 'signal-on' aptamer-based fluorescent probe platform for NEK9 imaging.
• Demonstrated successful visualization of intracellular NEK9 in live cells with minimal cytotoxicity (cell viability > 95%).

Conclusions:

• The developed aptamer-based fluorescent probe provides a robust tool for studying NEK9 kinase and its associated signaling pathways.
• This work establishes a generalizable strategy for overcoming key bottlenecks in live-cell imaging through rational aptamer engineering.