Enhancing Catalytic Activity of Uranyl-Dependent DNAzyme by Flexible Linker Insertion for More Sensitive Detection of Uranyl Ion

The uranyl-dependent DNAzyme 39E cleaves its nucleic acid substrate in the presence of uranyl ion (UO₂²⁺). It has been widely utilized in many sensor designs for selective and sensitive detection of UO₂²⁺ in the environment and inside live cells. In this work, by inserting a flexible linker (C3 Spacer) into one critical site (A₂₀) of the 39E catalytic core, we successfully enhanced the original catalytic activity of 39E up to 8.1-fold at low UO₂²⁺ concentrations. Applying such a modified DNAzyme (39E-A₂₀-C3) in a label-free fluorescent sensor for UO₂²⁺ detection achieved more than 1 order of magnitude sensitivity enhancement over using native 39E, with the UO₂²⁺ detection limit improved from 2.6 nM (0.63 ppb) to 0.19 nM (0.047 ppb), while the high selectivity to UO₂²⁺ over other metal ions was fully preserved. The method was also successfully applied for the detection of UO₂²⁺-spiked environmental water samples to demonstrate its practical usefulness.