Self-quenching smart probes as a platform for the detection of sequence-specific UV-induced DNA photodamage |
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Authors: | Sulayman A Oladepo Glen R Loppnow |
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Institution: | (1) Department of Chemistry, University of Alberta, Edmonton, AB, T6G 2G2, Canada;(2) Present address: Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA; |
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Abstract: | 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. |
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