Ultrasensitive, label-free detection of T4 ligase and T4 polynucleotide kinase based on target-triggered hyper-branched rolling circle amplification

•A simple but sensitive sensor is established for ligase and polynucleotide kinase (PNK) assay.•Exponential HRCA confers the sensor with high sensitivity and selectivity.•Label-free detection mode greatly simplifies the sensor design and reduces the cost.•The sensor was successfully used for ultrasensitive assay of T4 ligase and T4 PNK.•Such a sensing strategy can be easily extended to studies on enzyme inhibitors. Ligase and polynucleotide kinase (PNK) play important roles in DNA repair process. In this work, a simple, convenient and ultrasensitive fluorescence biosensor for label-free detection of ligase and PNK was proposed on the basis of target-triggered hyper-branched rolling circle amplification (HRCA). In the presence of PNK and ligase, the 5′-OH end of a linear Padlock DNA strand could be phosphorylated by PNK and then be converted by ligase into a circular template to trigger subsequent exponential HRCA reaction, producing amounts of double-stranded DNA (dsDNA) fragments, accompanied by the significant fluorescence increase of dsDNA-intercalating dye–SYBR Green I (SG I). In the absence of ligase or PNK, however, HRCA primer and uncyclized Padlock strands were completely digested by exonucleases, thus providing a low background for the sensing system. Therefore, by recording the fluorescence change of SG I, ligase and PNK activity could be facilely determined. The proposed biosensor exhibited excellent detection sensitivity for ligase and PNK activity with the detection limits of 3.4 × 10−4 U/mL and 3.8 × 10−4 U/mL, respectively. Such a sensing strategy could be easily extended to studies on inhibitors of these two enzymes, thus might offer a promising tool in clinical molecular diagnostics and cancer therapy.