In Vitro Selection of Hg (II) and As (V)-Dependent RNA-Cleaving DNAzymes

Abstract

DNAzymes (or catalytic DNA) are cell-free biomolecular recognition tools with target recognition sequences for charged molecules such as metal ions, antibiotics, and pharmaceuticals. In this study, using in vitro selection, large populations (e.g., 1015) of random DNA sequences were used as the raw material for the selection of "catalytic or functional molecules" for Hg2+ and As5+. From a random pool of 45-nt (Pool-A) and 35-nt (Pool-B) templates, we isolated RNA-cleaving catalytic Hg2+- and As5+-active DNAzymes, respectively. After eight cycles of selection and amplification wihin Pool A, sequences were enriched with a 54% cleavage efficiency against Hg2+. Similarly, Pool-B was found to catalyze ca. 18% cleavage efficiency against As5+ after 10 cycles of repeated selection and amplification. The M-fold software analysis resulted in sequences in the two active pools being dominated by "AATTCCGTAGGTCCAGTG" and "ATCTCCTCCTGTTC" functional motifs for Hg2+- and As5+-based catalysis, respectively. These DNAzymes were found to have higher activity in the presence of transition metal ions compared to alkaline earth metal ions. A maximum cleavage rate of 2.7 min−1 for Hg2+ was found to be highest in our study at a saturating concentration of 500 µM. The results demonstrate that DNAzymes are capable of selectively binding transition metal ions, and catalytic rates are at par with most Mg2+-dependent nucleic acid enzymes under similar conditions, and indicate their potential as metal-species specific biosensors.