Amplified detection of DNA ligase and polynucleotide kinase/phosphatase on the basis of enrichment of catalytic G-quadruplex DNAzyme by rolling circle amplification

• Two label-free G-quadruplex DNAzyme-based sensors were developed for T4 DNA ligase and T4 PNKP.
• Introduction of rolling circle amplification reaction significantly increased the sensitivities of the sensors.
• As low as 0.0019 and 0.0018 U/mL T4 DNA ligase and T4 PNKP could be detected respectively.
• The proposed sensors can also be extended to the studies on inhibitors of these two enzymes.

As two commonly used tool enzymes, DNA ligase and polynucleotide kinase/phosphatase (PNKP) play important roles in DNA metabolism. More and more studies show that regulation of their activity represents promising means for cancer therapy. To detect the activity of DNA ligase with high sensitivity and specificity, a G-quadruplex DNAzyme-based DNA ligase sensor was developed. In this sensor, the use of G-quadruplex DNAzyme eliminated the needs for any labeled oligonucleotide probes, thus making label-free detection possible. The introduction of rolling circle amplification (RCA) reaction could lead to the formation of multimeric G-quadruplexes containing thousands of G-quadruplex units, which can provide highly active hemin-binding sites, thus significantly improving the sensitivity of the sensor. The proposed sensor allowed specific detection of T4 DNA ligase with a detection limit of 0.0019 U/mL. By adding a PNKP-triggered 5′-phosphroylation step of the template DNA, the above sensing strategy could be easily extended to the design of PNKP sensor. The established sensor allowed specific detection of T4 PNKP with a detection limit of 0.0018 U/mL. In addition, these two sensors could also be used for the studies on inhibitors of these two enzymes.