Pb²⁺ as a Substrate and a Cofactor of a Porphyrin Metalation DNAzyme

We herein report a DNAzyme named T30695 (sequence: (G₃ T)₄ ) that can catalyze Zn²⁺ insertion into three different porphyrins in the presence of Pb²⁺ as a cofactor. Meanwhile, T30695 with Pb²⁺ alone was found to cause a shift in both the fluorescence and UV-vis spectra of protoporphyrin IX (PPIX), thus suggesting that metalation of Pb²⁺ was also achieved at room temperature. From kinetic measurements, the reaction required two Pb²⁺ ions; this is consistent with one being a cofactor and the other being a substrate. No previous reports inserted Pb²⁺ into porphyrins by using DNAzymes or protein-based enzymes. This reaction was most significantly inhibited in the presence of K⁺ followed by Na⁺ and Li⁺ , suggesting the importance of the Pb²⁺ -stabilized G-quadruplex. When Pb²⁺ is inserted into PPIX, its emission blue shifts from 635 to 590 nm, thus allowing simple ratiometric fluorescent sensing with a detection limit of 1.2 nM Pb²⁺ .