A label-free fluorescent biosensor for amplified detection of T4 polynucleotide kinase activity based on rolling circle amplification and catalytic hairpin assembly

T4 polynucleotide kinase (PNK) plays a key role in maintaining genome integrity and repairing DNA damage. In this paper, we proposed a label-free fluorescent biosensor for amplified detection of T4 PNK activity based on rolling circle amplification (RCA) and catalytic hairpin assembly (CHA). Firstly, we designed a padlock probe with a 5 '-hydroxyl terminus for phosphorylation reaction, a complementary sequence of the primer for initiating RCA, and a complementary sequence of the trigger for triggering CHA. T4 PNK catalyzed the phosphorylation reaction by adding a phosphate group to the 5 '-hydroxyl terminus of padlock probe, generating a phosphorylated padlock probe. Then it hybridized with the primer to generate a circular probe under the action of ligase. Subsequently, the primer initiated an RCA reaction along the circular probe to synthesize a large molecular weight product with repetitive trigger sequences. The triggers then triggered the cyclic assembly reactions be-tween hairpin probe 1 and hairpin probe 2 to generate a large amount of complexes with free G-rich sequences. The free G-rich sequences folded into G-quadruplex structures, and the N-methylmesoporphyrin IXs were inserted into them to produce an amplified fluorescent signal. Benefiting from high amplification efficiency of RCA and CHA, this fluorescent biosensor could detect T4 PNK as low as 6.63 x 10-4 U mL-1, and was suc-cessfully applied to detect its activity in HeLa cell lysates. Moreover, this fluorescent biosensor could effectively distinguish T4 PNK from other alternatives and evaluate the inhibitory effect of inhibitor, indicating that it had great potential in drug screening and disease treatment.

Automated summary

This paper presents a label-free fluorescent biosensor for amplified detection of T4 polynucleotide kinase (PNK) activity. The biosensor combines rolling circle amplification (RCA) and catalytic hairpin assembly (CHA) to achieve high amplification efficiency and can detect T4 PNK as low as 6.63 x 10-4 U mL-1. The biosensor has been successfully applied to detect T4 PNK activity in cell lysates and shows great potential in drug screening and disease treatment.