Secondary-Structure Characterization of Two Proficient Kinase Deoxyribozymes

Abstract

Dk1 and Dk2 are two catalytically proficient, manganese-dependent, guanine-rich deoxyribozymes previously isolated for DNA phosphorylation. In this study, we carried out a series of experiments that aimed to understand the structural properties of Dk1 and Dk2 and compare the structural similarities or differences of these two distinct deoxyribozymes that carry out similar catalytic functions. First, we performed reselections from two partially randomized DNA libraries on the basis of the original Dk1 and Dk2 sequences to isolate catalytically active sequence variants and identify nucleotides that are invariable, well-conserved, or highly mutagenized. Sequence analysis of these variants assisted by secondary-structure predictions led to the identification of possible Watson-Crick base-pairing regions within each deoxyribozyme. Sequence truncation and base-pair partner exchange experiments were conducted to confirm, or rule out, the existence of the predicted secondary-structure elements. Finally, methylation interference experiments were applied to identify nucleotides that are potentially important for the tertiary structure folding of the deoxyribozymes. Our data suggest that Dk1 and Dk2, despite the differences in their primary sequences and NTP requirements, use an analogous stem-loop element to anchor a structural domain of substantial tertiary interactions to execute their catalytic functions.

A Complex RNA-Cleaving DNAzyme That Can Efficiently Cleave a Pyrimidine–Pyrimidine Junction

“…Not surprisingly, all kinase DNAzymes reported to date are large (containing more than 40 nt) and exhibit complex secondary structures. 5,35,36,39 Interestingly, CT10-3.29 employs a structural arrangement that is quite similar to that of pH6DZ1, a fluorescence-signaling DNAzyme reported by our group for catalyzing the cleavage of an RNA linkage embedded in a DNA chain and flanked by 2 nt modified with bulky chromophores. 40 Both CT10-3.29 and pH6DZ1 adopt a four-way junction framework of comparable sizes with two intramolecular hairpin structures.…”

“…S3). In order to determine whether the N7 atoms of these guanine residues are involved in structural formation or catalysis, we conducted methylation interference assay with dimethyl sulfate (DMS) using a protocol previously described by our group 39,40 (the data are provided as Fig. S4 in Supplementary Information).…”

“…39,40 Five hundred picomoles of DNAzyme was radiolabeled with trace amounts of [γ 32 P]ATP and PNK for 20 min at 37°C. The solution was chased with nonradiolabeled ATP for 10 min and subsequently inactivated by heating at 90°C for 5 min.…”

Assembly and activation of a kinase ribozyme

“…The recent discovery of metabolite-linked and cofactor-linked RNAs in cell extracts Kowtoniuk et al 2009) further raises the possibility that ribozymes may have important roles in metabolic transformations of small molecules in modern biology. Phosphoryl transfer is of special interest because of its ubiquitous role in cellular biology, and phosphoryl transfer ribozymes (and deoxyribozymes) have been isolated and described by us (Rhee and Burke 2004;Saran et al 2005Saran et al , 2006Biondi et al 2010) and by others Szostak 1994, 1995;Li and Breaker 1999;Wang et al 2002;Achenbach et al 2005;Curtis and Bartel 2005;Chiuman and Li 2006;Li 2007, 2008). Nevertheless, there is still relatively little known about the mechanisms by which nucleic acids catalyze phosphoryl transfer or the structures that they use to do so.…”

A Deoxyribozyme with a Novel Guanine Quartet-Helix Pseudoknot Structure

“…We used a family of self-phosphorylating DNAzymes (or kinase DNAzymes) as a model system to study the capacity of single-stranded DNA to form diverse and complex structures. [11][12][13] Although highresolution study of DNAzymes has remained elusive, we can use a series of biochemical experiments to deduce many structural elements of a DNAzyme to create a detailed structural model. Here we report the characterization of a selfphosphorylating DNAzyme that reveals a new structural arrangement containing a helical element interacting with a two-tiered G-quadruplex.…”