In Vitro Selection of Hg (II) and As (V)-Dependent RNA-Cleaving DNAzymes
Environmental Engineering Science
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 15 ) of random DNA sequences were used as the raw material for the selection of "catalytic or functional molecules" for Hg 2ϩ and As 5ϩ . From a random pool of 45-nt (Pool-A) and 35-nt (Pool-B) templates, we isolated RNA-cleaving catalytic Hg 2ϩ
... nd As 5ϩ -active DNAzymes, respectively. After eight cycles of selection and amplification wihin Pool A, sequences were enriched with a 54% cleavage efficiency against Hg 2ϩ . Similarly, Pool-B was found to catalyze ca. 18% cleavage efficiency against As 5ϩ after 10 cycles of repeated selection and amplification. The M-fold software analysis resulted in sequences in the two active pools being dominated by "AATTCCGTAG-GTCCAGTG" and "ATCTCCTCCTGTTC" functional motifs for Hg 2ϩ -and As 5ϩ -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 Ϫ1 for Hg 2ϩ was found to be highest in our study at a saturating concentration of 500 M. The results demonstrate that DNAzymes are capable of selectively binding transition metal ions, and catalytic rates are at par with most Mg 2ϩ -dependent nucleic acid enzymes under similar conditions, and indicate their potential as metal-species specific biosensors.