In vitro selection of deoxyribozymes active with Cd²⁺ ions resulting in variants of DNAzyme 8–17

Abstract

In vitro selection was performed to search for RNA-cleaving DNAzymes catalytically active with Cd(2+) ions from the oligonucleotide combinatorial library with a 23-nucleotide random region. All the selected, catalytically active variants turned out to belong to the 8-17 type DNAzyme. Three DNAzymes were prepared in shortened, cis-acting versions which were subjected to a detailed study of the kinetic properties and metal ion preferences. Although the selection protocol was designed for Cd(2+)-dependent DNAzymes, the variants showed broader metal ion specificity. They preferred Cd(2+) but were also active with Mn(2+) and Zn(2+), suggesting that binding of the catalytic ion does not require an extremely specific coordination pattern. The unexpected decrease of the catalytic activity of the variants along with the temperature increase suggested that some changes occurred in their structures or the rate-limiting step of the reaction was changed. Two elements of the catalytic core of DNAzyme 1/VIIWS, the nucleotide at position 12 and the three-base-pair hairpin motif, were mutated. The presence of a purine residue at position 12 was crucial for the catalytic activity but the changes at that position had a relatively small influence on the metal ion preferences of this variant. The middle base pair of the three-base-pair hairpin was changed from A-T to C-G interaction. The catalytic activity of the mutated variant was increased with Zn(2+), decreased with Mn(2+), and was not changed in the presence of Cd(2+) ions. Clearly, this base pair was important for defining the metal ion preferences of the DNAzyme 1/VIIWS.

Theranostic DNAzymes

“…Many interesting progresses were made by using transition metal ions 7, 11, 35. Many DNAzymes were reported to use Zn 2+ 26, 36, UO 2 2+ 14, Cu 2+ 37, Cd 2+ 29, 38, Hg 2+ 39, and Ag + 40. We also isolated a series of lanthanide-dependent DNAzymes 15, 41-43.…”

“…We reported that the 8-17 is even faster than the 10-23 for cleaving chimeric substrates in the presence of Mg 2+ 25. The 8-17 motif has been repeated selected by several labs using Zn 2+ 26, Mg 2+ 27, 28, Ca 2+ 17, 28, Cd 2+ 29, and other conditions with the best activity observed with Pb 2+ 30. Its recurrence has been attributed to the small size, high tolerance to mutations, and high activity 27.…”

In Vitro Selection in Serum: RNA-Cleaving DNAzymes for Measuring Ca2+ and Mg2+

“…6 Therefore, it is not surprising that variants of 17E were selected here. In fact, 17E variants have been isolated 8 from many previous selections using different metal ions, 20,22,[32][33][34] which is attributed to its small size, tolerance to mutation, and high activity. 35,36 Unlike previous selections done in clean buffers, serum contains many other chemicals (e.g.…”

“…It was previously reported that with this position being an adenine, the activity is optimal with Cd 2+ , Zn 2+ and Mn 2+ . 34 Figure S4). Earlier studies pointed out that the N12 position plays two crucial roles in 17E.…”

“…33 Similar conclusions were also made by Kasprowicz et al in a recent report. 34 With our observed metal preference in these 17E variants, we next aimed to test the feasibility of using these two related DNAzymes for the analysis of Ca 2+ and Mg 2+ , which are two important physiological metal ions. Note that 17EV1 and 17E are also different in the stem region.…”