In Vitro Selection of a DNAzyme Cooperatively Binding Two Lanthanide Ions for RNA Cleavage
2016
Huang, Po-Jung Jimmy | Vazin, Mahsa | Liu, Juewen
Trivalent lanthanide ions (Ln³⁺) were recently employed to select RNA-cleaving DNAzymes, and three new DNAzymes have been reported so far. In this work, dysprosium (Dy³⁺) was used with a library containing 50 random nucleotides. After six rounds of in vitro selection, a new DNAzyme named Dy10a was obtained and characterized. Dy10a has a bulged hairpin structure cleaving a RNA/DNA chimeric substrate. Dy10a is highly active in the presence of the five Ln³⁺ ions in the middle of the lanthanide series (Sm³⁺, Eu³⁺, Gd³⁺, Tb³⁺, and Dy³⁺), while its activity descends on the two sides. The cleavage rate reaches 0.6 min–¹ at pH 6 with just 200 nM Sm³⁺, which is the fastest among all known Ln³⁺-dependent enzymes. Dy10a binds two Ln³⁺ ions cooperatively. When a phosphorothioate (PS) modification is introduced at the cleavage junction, the activity decreases by >2500-fold for both the Rₚ and Sₚ diastereomers, and thiophilic Cd²⁺ cannot rescue the activity. The pH–rate profile has a slope of 0.37 between pH 4.2 and 5.2, and the slope was even lower at higher pH. On the basis of these data, a model of metal binding is proposed. Finally, a catalytic beacon sensor that can detect Ho³⁺ down to 1.7 nM is constructed.
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