Fluorescent single-stranded DNA-binding protein from Plasmodium falciparum as a biosensor for single-stranded DNA

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Single-stranded DNA (ssDNA) is transiently formed during crucial cellular processes like DNA replication and repair.
• Measuring ssDNA formation is vital for understanding these fundamental biological mechanisms.
• Existing biosensors, like DCC-EcSSB, faced limitations due to complex binding modes affecting ssDNA quantitation.

Purpose of the Study:

• To develop an improved fluorescent biosensor for sensitive and accurate detection of ssDNA.
• To utilize a novel scaffold, tetrameric SSB from Plasmodium falciparum, for enhanced biosensor performance.
• To enable simple and reliable quantitation of ssDNA in various cellular contexts.

Main Methods:

• Engineered a tetrameric SSB from Plasmodium falciparum, labeling each subunit with a diethylaminocoumarin fluorophore at a single surface site.
• Characterized the fluorescent properties of the labeled SSB (DCC-PfSSB), observing a 20-fold fluorescence increase upon ssDNA binding.
• Assessed the binding kinetics and affinity, determining a dissociation constant <5 pM for DCC-PfSSB.

Main Results:

• The DCC-PfSSB biosensor exhibits a significant fluorescence enhancement upon binding to ssDNA, indicating its utility.
• PfSSB demonstrates a consistent binding mode (65-70 bases per tetramer) across conditions, simplifying data interpretation.
• The biosensor was successfully applied in real-time assays, exemplified by measuring AddAB helicase activity during double-stranded DNA unwinding.

Conclusions:

• The novel DCC-PfSSB biosensor offers a significant improvement for ssDNA detection and quantitation.
• Its simple binding mode and high affinity make it ideal for real-time monitoring of cellular processes involving ssDNA.
• This tool facilitates a deeper understanding of DNA metabolism and related cellular functions.