Protocol for assembling photostable fluorescence-labeled tiny DNA origami blocks to observe kinesin cooperation

Area of Science:

• Biophysics
• Molecular Biology
• Nanotechnology

Background:

• Kinesin motor proteins are crucial for intracellular transport.
• Observing kinesin cooperation at the single-molecule level is challenging due to limitations like blinking and photobleaching.
• Novel tools are needed to visualize motor protein dynamics with high fidelity.

Purpose of the Study:

• To develop a protocol for assembling fluorescence-labeled tiny DNA origami blocks (FTOBs).
• To enable the observation of kinesin motility and their cooperative behaviors.
• To provide a versatile method applicable to various motor proteins.

Main Methods:

• Assembly of FTOBs using DNA origami techniques.
• Labeling FTOBs with kinesin motor proteins via an ALFA-tag and nanobody (NbALFA) system.
• Connecting FTOB-kinesin complexes using complementary DNA arms for hybridization.
• Single-molecule tracking of the assembled complexes.

Main Results:

• Successfully assembled FTOBs for kinesin observation.
• Demonstrated minimal blinking and photobleaching during single-molecule tracking.
• Enabled visualization of kinesin cooperation dynamics.
• Established a robust protocol for motor protein studies.

Conclusions:

• The developed FTOB protocol offers a powerful tool for studying motor protein dynamics.
• This method significantly advances the ability to observe kinesin cooperation.
• The protocol's versatility makes it applicable to a broad range of motor protein research.