In Vitro Selection of Mercury (II)- and Arsenic (V)-Dependent RNA-Cleaving DNAzymes

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., 10) of random DNA sequences were used as the raw material for the selection of ‘‘catalytic or functional molecules’’ for Hg2þ and As5þ. From a random pool of 45-nt (Pool-A) and 35-nt (Pool-B) templates, we isolated RNA-cleaving catalytic Hg2þand As5þactive DNAzymes, respectively. After eight cycles of selection and amplification wihin Pool A, sequences were enriched with a 54% cleavage efficiency against Hg2þ. Similarly, Pool B was found to catalyze ca. 18% cleavage efficiency against As5þ 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 Hg2þand As5þ-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 Hg2þ was found to be highest in our study at a saturating concentration of 500 mM. Results demonstrate that DNAzymes are capable of selectively binding transition metal ions, and catalytic rates are at par with most Mg2þ-dependent nucleic acid enzymes under similar conditions, and indicate their potential as metal species-specific biosensors.