Efficient DNA-Catalyzed Porphyrin Metalation for Fluorescent Ratiometric Pb²⁺ Detection

Developing biosensors for Pb²⁺ is an important analytical topic. DNA-based Pb²⁺ sensors have been designed mainly based on RNA-cleaving DNAzymes and Pb²⁺-induced folding of G-quadruplex (G4) DNA. Porphyrin metalation is a key reaction in biology and catalysis. Many enzyme mimics have been developed to catalyze this reaction, and some metalation DNAzymes were reported with a G4 structure. Inspired by the excellent G4 binding properties of certain divalent metal ions, we herein screened a few metals and G-rich DNA sequences. The metalation activity of a DNA named T30695 (sequence: (G₃T)₄) was significantly accelerated by Pb²⁺. The reaction of Cu²⁺ insertion into the mesoporphyrin IX had a k_cat of 0.89 min^-1 and a K_m of 9.8 μM, representing a catalytic efficiency similar to that of human ferrochelatase. The reason for the acceleration was attributed to Pb²⁺ binding of the G4 DNA and the catalytic activity of the large Pb²⁺ ion for this reaction. A ratiometric sensor for Pb²⁺ was developed by inserting Zn²⁺ with a detection limit of 23.5 nM Pb²⁺. This work has established a new DNA-based reaction that can be used for Pb²⁺ detection, and it also provides a highly efficient new DNAzyme for porphyrin metalation, which might be used for signal production for other biosensors.