Cleavable Substrate Containing Molecular Beacons for the Quantification of DNA-Photolyase Activity

In order to gain deeper insight into the function and interplay of proteins in cells it is essential to develop methods that allow the profiling of protein function in real time, in solution, in cells, and in cell organelles. Here we report the development of a U‐type oligonucleotide (molecular beacon) that contains a fluorophore and a quencher at the tips, and in addition a substrate analogue in the loop structure. This substrate analogue induces a hairpin cleavage in response to enzyme action, which is translated into a fluorescence signal. The molecular beacon developed here was used to characterize DNA‐photolyase activity. These enzymes represent a challenge for analytical methods because of their low abundance in cells. The molecular beacon made it possible to measure the activity of purified class I and class II photolyases. Photolyase activity was even detectable in crude cell extracts. Stressed out? A high‐throughput assay has been developed for analysis of the UV stress tolerance of organisms. It is based on a molecular beacon containing an activity‐translating substrate analogue in the head region (see scheme). The substrate analogue converts photolyase activity into cleavage of a hairpin DNA strand, which can be observed by use of the fluorescent beacon. Rapid activity profiling was achieved and photolyase activity could even be monitored in real time in crude cell extracts.