GR5

Lead (Pb) is a highly toxic heavy metal of great environmental and health concerns, and interestingly Pb2+ has played important roles in nucleic acids chemistry. Since 2000, using DNA for selective detection of Pb2+ has become a rapidly growing topic in the analytical community. Pb2+ can serve as the most active cofactor for RNA-cleaving DNAzymes including the GR5, 17E and 8-17 DNAzymes. Recently, Pb2+ was found to promote a porphyrin metalation DNAzyme named T30695. In addition, Pb2+ can tightly bind to various G-quadruplex sequences inducing their unique folding and binding to other molecules such as dyes and hemin. The peroxidase-like activity of G-quadruplex/hemin complexes was also used for Pb2+ sensing. In this article, these Pb2+ recognition mechanisms are reviewed from fundamental chemistry to the design of fluorescent, colorimetric, and electrochemical biosensors. In addition, various signal amplification mechanisms such as rolling circle amplification, hairpin hybridization chain reaction and nuclease-assisted methods are coupled to these sensing methods to drive up sensitivity. We mainly cover recent examples published since 2015. In the end, some practical aspects of these sensors and future research opportunities are discussed.

Lead contamination has posed a potential threat to the environment and food safety, arousing extensive concern. In this work, we fabricated a novel fluorescent sensing platform based on zeolitic imidazolate framework-8 (ZIF-8) and DNAzyme for monitoring Pb2+ in water and fish samples. ZIF-8 was proposed as a fluorescence quencher with the advantages of simple synthesis, low cost, and high quenching efficiency. The Pb2+-dependent GR5 DNAzyme containing the large ssDNA loop can be adsorbed onto ZIF-8 accompanied by fluorescence quenching. Upon binding with Pb2+, GR5 DNAzyme was activated and cleaved, leading to the release of FAM-labeled 5-base ssDNA, which restored the fluorescence. The "turn-on" assay can detect Pb2+ through the one-pot procedure in the range of 0.01-10.0 nM with a detection limit of 7.1 pM. The platform is promising for on-site monitoring of Pb2+ owing to the excellent performance of high sensitivity, low background, strong anti-interference ability, and simple operation.

In 1994, the first DNAzyme named GR5 was reported, which specifically requires Pb2+ for its RNA cleavage activity. Three years later, the 8-17 DNAzyme was isolated. The 8-17 DNAzyme and the related 17E DNAzyme are also most active with Pb2+ , although other divalent metals can work as well. GR5 and 17E have the same substrate sequence, and their catalytic loops in the enzyme strands also have a few similar and conserved nucleotides. Considering these, we hypothesized that 17E might be a special form of GR5. To test this hypothesis, we performed systematic rational evolution experiments to gradually mutate GR5 toward 17E. By using the activity ratio in the presence of Pb2+ and Mg2+ for defining these two DNAzymes, the critical nucleotide was identified to be T12 in 17E for metal specificity. In addition, G9 in GR5 is a position not found in most 17E or 8-17 DNAzymes, and G9 needs to be added to rescue GR5 activity if T12 becomes a cytosine. This study highlights the links between these two classic and widely used DNAzymes, and offers new insight into the sequence-activity relationship related to metal selectivity.