Label-Free Fluorescent Detection of T4 PNK Using a DNA Mimic Green Fluorescent Protein-Based Turn-On Biosensor

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• T4 polynucleotide kinase (T4 PNK) plays a crucial role in DNA metabolism and repair.
• Accurate detection of T4 PNK activity is essential for understanding nucleic acid metabolism and for biomedical applications.
• Existing methods for T4 PNK detection often lack sensitivity, specificity, or require labeling.

Purpose of the Study:

• To develop a novel, label-free, turn-on fluorescent biosensor for sensitive and specific detection of T4 PNK activity.
• To integrate DNA mimic green fluorescent protein (DMGFP) with λ exonuclease (λ exo) cleavage for T4 PNK detection.
• To demonstrate the biosensor's utility in evaluating enzyme inhibition and quantifying endogenous PNK activity.

Main Methods:

• Utilized T4 PNK-catalyzed phosphorylation of hairpin DNA (HP-DNA1) to initiate a cascade reaction.
• Employed λ exonuclease (λ exo) for sequential digestion of HP-DNA1, releasing single-stranded DNA (ssDNA1).
• Assembled an active DMGFP structure through hybridization of ssDNA1 with DNA2, leading to fluorescence enhancement upon DFHBI-1T binding.

Main Results:

• The biosensor demonstrated a linear response for T4 PNK activity from 0 to 0.5 U/mL.
• Achieved a low detection limit of 0.01 U/mL with outstanding specificity for T4 PNK.
• Successfully quantified endogenous polynucleotide kinase phosphatase (PNKP) in HeLa cell lysates and evaluated inhibitor concentrations.

Conclusions:

• The developed DMGFP-based fluorescent biosensor provides a reliable and versatile platform for T4 PNK activity analysis.
• The label-free, rapid-response, and low-background characteristics make it suitable for inhibitor screening and clinical diagnosis.
• This strategy holds significant potential for advancing nucleic acid metabolism research and biomedical applications.