TtAgo sensor for the sensitive and rapid detection of T4 polynucleotide kinase activity

As a next-generation biosensing technology, the application of Argonaute for analytical science research has attracted considerable attention. A Thermus thermophilus Argonaute (TtAgo)-based sensor is presented for detecting T4 polynucleotide kinase (PNK) activity considering that TtAgo's multiple-turnover catalytic activity can be activated by binding to a 5 '-phosphate termini of single-stranded DNA (ssDNA) for the cleavage of complementary strand. PNK catalyzes the phosphorylation of 5 '-hydroxyl termini of ssDNA, and the phosphor-ylation product termed guide can activate the TtAgo-mediated complementary ssDNA reporter cleavage to release fluorescence signal. For the PNK activity monitoring, the developed method shows good analytic per-formance with a linear range of 0.0010-2 U mL-1 and a detection limit of 0.0010 U mL-1 without the use of a preamplification reaction. This method is successfully applied for the analysis of HeLa cells at a single-cell level. The developed method shows several advantages, including high sensitivity, low cost, facile operation, and has remarkable potential application for early disease diagnosis, drug screening, and clinical treatment.