Site-Selective Labeling of Chromium(III) as a Quencher on DNA for Molecular Beacons

Molecular beacons typically use organic molecules or nanomaterials as quenchers. Many transition-metal ions have excellent fluorescence quenching ability, and the aim of this study was to recruit them as small quenchers in DNA detection. Cr³⁺ has a slow ligand exchange rate, forming stable adducts with DNA. With its strong fluorescence quenching ability, the site-specific labeling of Cr³⁺ on DNA to form a new type of molecular beacon was investigated. The kinetics of quenching by Cr³⁺ were measured for single- and double-stranded DNA as a function of salt concentration, pH, and Cr³⁺ ion concentration. The goal was to achieve a selective reaction with the single-stranded but not double-stranded regions. The reaction mechanism was also probed by adding adenosine triphosphate, revealing two Cr³⁺ -binding modes: fast but unstable, and slow but stable. A partially complementary duplex was designed with a short polyguanine overhang, which, under optimal conditions, enabled selective labeling of the overhanging region with Cr³⁺ . The resulting sequence was tested as a molecular beacon with a detection limit of 0.3 nm DNA and a saturated fluorescence enhancement of fivefold. With a 13-nucleotide target DNA, the single mismatch discrimination of the beacon was 22-fold. This study demonstrates the possibility of forming useful Cr³⁺ adducts with DNA. Such adducts are not only useful for developing biosensors but also for constructing new materials.