Self-quenching smart probes as a platform for the detection of sequence-specific UV-induced DNA photodamage

摘要

Molecular beacons (MBs) are sensitive probes for many DNA sequence-specific applications, such as DNA damage detection, but suffer from technical and cost limitations. We have designed smart probes with self-quenching properties as an alternative to molecular beacons to monitor sequence-specific UV-induced photodamage of oligonucleotides. These probes have similar stem-loop structural characteristics as molecular beacons, but quenching is achieved instead via photoinduced intramolecular electron transfer by neighboring guanosine residues. Our results indicate that the probes are sensitive enough to detect nanomolar target concentrations and are specific enough to discriminate single-base damage. When the probes were used to monitor UV-induced photodamage in oligonucleotide sequences that differ by a single-base mismatch, the photodamage time constant was higher for the perfectly complementary target sequences than for the mismatch sequences, indicating that these probes are specific for each target sequence. In addition, time constants obtained for oligonucleotide target sequences with both stem and loop base mismatches are lower than those with only loop mismatches, suggesting that these sequences are also specifically distinguished by the smart probes. These probes thus constitute robust, sensitive, specific, and cheaper alternatives to MBs for sequence-specific DNA damage detection.