Abstract:

Cu2+ is a very important metal ion in biology, environmental science, and industry. Developing biosensors for Cu2+ is a key topic in analytical chemistry. DNAzyme-based sensors are highly attractive for their excellent sensitivity, stability, and programmability. In the past decade, a few Cu2+ biosensors were reported using DNAzymes with DNA cleavage or DNA ligation activity. However, they require unstable ascorbate or imidazole activation. So far, no RNA-cleaving DNAzymes specific for Cu2+ are known. In this work, a phosphorothioate (PS) RNA-containing library was used for in vitro selection, and a few new Cu2+-specific RNA-cleaving DNAzymes were isolated. Among them, a DNAzyme named PSCu10 was studied further. It has only eight nucleotides in the enzyme loop with a cleavage rate of 0.1 min–1 in the presence of 1 μM Cu2+ at pH 6.0 (its optimal pH). Between the two diastereomers of the PS RNA chiral center, the Rp isomer is 37 times more active than the Sp one. Among the other divalent metal ions, only Hg2+ can cleave the substrate due to its extremely high thiophilicity. A catalytic beacon sensor was designed with a detection limit of 1.6 nM Cu2+ and extremely high selectivity. PSCu10 is specific for Cu2+, and it has no cleavage in the presence of ascorbate, which reduces Cu2+ to Cu+.