Direct observation method of individual single-stranded DNA molecules using fluorescent replication protein A

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Single-molecule studies offer unique insights into DNA metabolism, surpassing bulk experiment limitations.
• Observing single DNA molecules requires specific labeling techniques, with challenges in visualizing single-stranded DNA (ssDNA).
• Current methods for double-stranded DNA (dsDNA) visualization are insufficient for differentiating ssDNA and dsDNA in complex reactions.

Purpose of the Study:

• To develop a method for direct observation and discrimination of ssDNA and dsDNA.
• To enable detailed analysis of DNA metabolism processes at the single-molecule level.
• To overcome limitations in existing fluorescent labeling techniques for ssDNA.

Main Methods:

• Engineered a fusion protein combining the DNA-binding domain of replication protein A (RPA) with enhanced yellow fluorescent protein (RPA-YFP).
• Verified the ssDNA-binding activity of the RPA-YFP fusion protein.
• Employed simultaneous staining: SYTOX Orange for dsDNA and RPA-YFP for ssDNA.

Main Results:

• The RPA-YFP fusion protein successfully binds to ssDNA.
• Achieved simultaneous and distinct visualization of ssDNA and dsDNA within the same sample.
• Demonstrated the utility of RPA-YFP in conjunction with SYTOX Orange for dual-labeling.

Conclusions:

• The developed RPA-YFP fusion protein provides a novel tool for direct ssDNA visualization.
• This method enables simultaneous discrimination between ssDNA and dsDNA, crucial for studying DNA metabolism.
• Facilitates advanced single-molecule studies of DNA replication, repair, and recombination.