Hairpin probes for real-time assay of restriction endonucleases

Abstract

New fluorogenic probes for restriction endonucleases based on molecular beacons were developed. These hairpin probes were single-stranded oligonucleotides that had stem-and-loop structure and carried 5'-fluorophor moiety and 3'-quencher moiety. Their stem sequences were designed as recognition sites for restriction endonucleases and loop sequences were unrelated nucleotides. Upon cleavage by endonuclease, these probes became fluorescent and thus could trace the enzyme activity continuously. Two probes were designed for BglII and NcoI, respectively, and each was labeled with a fluorophor of different color. The results showed that the two probes could specifically assay for the corresponding enzymes either individually or simultaneously in a real-time mode. Considering the simplicity, quantification and high throughput, these probes could be extended to other applications such as drug screening, protein-nucleic acid interaction study and searching for small molecule DNA cleavage agents.