Anchored linear oligonucleotides: the effective tool for the real-time measurement of uracil DNA glycosylase activity

Base excision repair is one of the important DNA repair mechanisms in cells. The fundamental role in this complex process is played by DNA glycosylases. Here, we present a novel approach for the real-time measurement of uracil DNA glycosylase activity, which employs selected oligonucleotides immobilized on the surface of magnetic nanoparticles and Forster resonance energy transfer. We also show that the approach can be performed by surface plasmon resonance sensor technology. We demonstrate that the immobilization of oligonucleotides provides much more reliable data than the free oligonucleotides including molecular beacons. Moreover, our results show that the method provides the possibility to address the relationship between the efficiency of uracil DNA glycosylase activity and the arrangement of the used oligonucleotide probes. For instance, the introduction of the nick into oligonucleotide containing the target base (uracil) resulted in the substantial decrease of uracil DNA glycosylase activity of both the bacterial glycosylase and glycosylases naturally present in nuclear lysates.

Automated summary

This study introduces a novel approach for real-time measurement of uracil DNA glycosylase activity by immobilizing selected oligonucleotides on magnetic nanoparticles and utilizing Forster resonance energy transfer. The results demonstrate that this method provides more reliable data than using free oligonucleotides, and offers the possibility to investigate the relationship between uracil DNA glycosylase activity and the arrangement of oligonucleotide probes. Additionally, introducing a nick into oligonucleotides containing the target base (uracil) significantly decreases the activity of both bacterial glycosylase and glycosylases naturally present in nuclear lysates.