Amplified detection of T4 polynucleotide kinase activity by the coupled λ exonuclease cleavage reaction and catalytic assembly of bimolecular beacons.

The phosphorylation of nucleic acid catalyzed by polynucleotide kinase is an indispensible procedure involved in many vital cellular activities such as DNA recombination and DNA repair. Herein, a novel strategy for the sensitive determination of T4 polynucleotide kinase (PNK) activity and inhibition was proposed, which combined exonuclease enzyme reaction and bimolecular beacons (bi-MBs)-based signal amplification. A hairpin probe (HP) with 5'-hydroxyl termini and two different types of molecular beacons (MBs), MB1 and MB2, is designed. Taking advantage of the efficient enzyme reactions, namely the phosphorylation of HP by PNK and the λ exonuclease cleavage reaction, the trigger DNA fragment can be released from HP and is used to trigger the catalytic assembly of bimolecular beacons, resulting in a remarkably amplified fluorescence signal toward PNK activity detection. The detection limit of this method toward PNK was obtained as 1 mU/mL, which was superior or comparable with the reported methods. Furthermore, the facile and sensitive method can also be used to screen the inhibition effects toward several common inhibitors. It provides a promising platform for sensitive determination of nucleotide kinase activity and inhibition, and also shows great potential for biological process research, drug discovery, and clinic diagnostics.