T4 polynucleotide kinase (PNK) plays critical roles in regulating DNA phosphorylation
modes during the repair of DNA lesions. The aberrant activity of T4 PNK has been proven
to be associated with a variety of human pathologies. Sensitive detection of T4 PNK
activity is critical to both clinical diagnosis and therapeutics. Herein, a background-eliminated
fluorescence assay for sensitive detection of T4 PNK activity has been developed by
multifunctional magnetic probes and polymerization nicking reactions mediated hyperbranched
rolling circle amplification (HRCA). First, the streptavidin-magnetic nanobeads (MBs)
were functionalized with the biotin modified hairpin probe (HP) with 3'-phosphoryl,
forming multifunctional magnetic probes (HP-MBs). Then, in the presence of T4 PNK,
the 3'-phosphoryl of HP-MBs was hydrolyzed to 3'-hydroxyl, thus serving as primers
to initiate the polymerization extension and nicking endonuclease cleavage reaction.
Next, the primers released from above "polymerization-nicking" cycles were separated
out to trigger the subsequently HRCA process, producing plenty of dsDNA. Finally,
the intercalating dye SYBR Green I (SG) was inserted into the dsDNA, generating enhanced
fluorescence signals. In our design, the HP-MBs here serve together as the T4 PNK,
DNA polymerase, and endonuclease recognition probe, and thus avoid the demands of
utilizing multiple probes design. Moreover, it performed primary "polymerization-nicking"
amplification and mediate secondary HRCA. In addition to, performing the separation
function, the binding of HP-MBs and SG could be avoided while a low background was
acquired. This method showed excellent sensitivity with a detection limit of 0.0436
mU/mL, and accomplished exceptional characterization T4 PNK activity in cell extracts,
offering a powerful tool for biomedical research and clinical diagnosis.