Primer-template conversion-based cascade signal amplification strategy for sensitive and accurate detection of polynucleotide kinase activity

Here, a primer-template conversion-based cascade signal amplification strategy is described for the sensitive detection of polynucleotide kinase (PNK) activity. This strategy integrated rolling circle amplification (RCA) and multiple-repeated-strand displacement amplification (MRSDA) with G-quadruplex based fluorescence lighting-up assay. A delicate dumbbell-shaped DNA probe with 5'-hydroxyl terminus was designed, in which G-quadruplex and half recognition site of nicking enzyme Nb.BbvCI were encoded in two loops respectively. Under the action of PNK, the 5' terminus on dumbbell probe was firstly phosphorylated, and then the dumbbell was cyclized with the catalyzation of T4 ligase to become the RCA template. The RCA process produced multiple copies of the prolonged primer. After that, under the assistance of nicking enzyme Nb.BbvCI, a primer-template conversion occurred, which converted the primer and template of RCA into the template and primer of the subsequent MRSDA, respectively. The MRSDA generated multiple repeated ssDNA sequences which possessed G-quadruplexes for outputting signal by lighting-up fluorescence of thioflavin T (ThT). The cascade signal amplification of RCA and MRSDA provided high detection sensitivity, and the target-dependence of template in cascade signal amplification led to a low background. The method showed excellent detection limit of 0.2 × 10-6 U μL-1 in buffer and 5 cells in cell lysate sample. Moreover, this method displayed favorable selectivity when interfering proteins were present. The developed strategy has good practical potential for PNK activity detection in clinical diagnosis and medical research.