Molecular Features and Metal Ions That Influence 10-23 DNAzyme Activity

Abstract

<jats:p>Deoxyribozymes (DNAzymes) with RNA hydrolysis activity have a tremendous potential as gene suppression agents for therapeutic applications. The most extensively studied representative is the 10-23 DNAzyme consisting of a catalytic loop and two substrate binding arms that can be designed to bind and cleave the RNA sequence of interest. The RNA substrate is cleaved between central purine and pyrimidine nucleotides. The activity of this DNAzyme in vitro is considerably higher than in vivo, which was suggested to be related to its divalent cation dependency. Understanding the mechanism of DNAzyme catalysis is hindered by the absence of structural information. Numerous biological studies, however, provide comprehensive insights into the role of particular deoxynucleotides and functional groups in DNAzymes. Here we provide an overview of the thermodynamic properties, the impact of nucleobase modifications within the catalytic loop, and the role of different metal ions in catalysis. We point out features that will be helpful in developing novel strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. Consideration of these features will enable to develop improved strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. These insights provide the basis for improving activity in cells and pave the way for developing DNAzyme applications.</jats:p>

Viroids and the Origin of Life

“…Additionally, DNA has been proposed to have arisen before a reverse transcriptase enzyme existed by chemical deoxygenation of ribonucleotides [ 3 ]. Similar functions of ribozymes have recently been attributed to desoxyribozymes, “DNAzymes”, which can cleave and join and show similar properties as RNA ribozymes [ 35 ].…”

A computational approach to identify efficient RNA cleaving 10–23 DNAzymes

“…For instance, in the computational scan of siRNA molecules there are additional factors that can be taken into account, such as internal structure of the target RNA ( 36 ) and specific nucleotide composition ( 37 ), among others [reviewed in ( 38 )]. A specific feature of DNAzymes that can also be considered is the preference of certain purine:pyrimidine junctions over other combinations ( 18 , 28 ). We specifically focused here on those junctions reported to be the most efficient (GU and AU), but future models may incorporate other combinations to study the joint effect of purine:pyrimidine junctions and other features.…”

“…Here, we focus on the ‘10–23’ DNAzyme, an RNA-cleaving molecule, consisting of a 15-nucleotide catalytic core flanked by substrate binding arms of variable length and sequence that confer specificity by interacting with target RNA via Watson–Crick base pairing interactions ( 16 ). These DNAzymes cleave RNA between an unpaired purine (A, G) and a paired pyrimidine (U, C) in the presence of a divalent cation, such as Mg 2+ , via a de-esterification reaction ( 17 , 18 ). This results in phosphodiester bond cleavage of the target and generation of two RNA fragments ( 16 ).…”

Specific DNAzymes cleave the 300–618 nt of 5′UTR to inhibit DHAV-1 translation and replication

“…Metal ions are essential elements for the catalytic activity of most known DNAzymes and play key roles in the catalytic process ( McGhee et al, 2017 ; Rosenbach et al, 2020 ). We found that DNAzymes showed different cleavage activity rates in the presence of divalent metal ions ( Figures 3D – F ), which may have been related to the inherent phosphate affinities of the different metal ions ( Schnabl and Sigel, 2010 ).…”