Small, Highly Active DNAs That Hydrolyze DNA

Abstract

DNA phosphoester bonds are exceedingly resistant to hydrolysis in the absence of chemical or enzymatic catalysts. This property is particularly important for organisms with large genomes, as resistance to hydrolytic degradation permits the long-term storage of genetic information. Here we report the creation and analysis of two classes of engineered deoxyribozymes that selectively and rapidly hydrolyze DNA. Members of class I deoxyribozymes carry a catalytic core composed of only 15 conserved nucleotides and attain an observed rate constant (kobs) of ~1 min−1 when incubated near neutral pH in the presence of Zn2+. Natural DNA sequences conforming to the class I consensus sequence and structure were found that undergo hydrolysis under selection conditions (2 mM Zn2+, pH 7), which demonstrates that the inherent structure of certain DNA regions might promote catalytic reactions leading to genomic instability.

Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes

“…These reactions include Cu 2+ -dependent oxidative cleavage [46–48], Cu 2+ /Mn 2+ -dependent oxidative nucleoside excision [49,50], DNA phosphodiester hydrolysis [51,52], RNA hydrolysis (involving a water molecule, a normally disfavored reaction relative to RNA cleavage by the Figure 1 mechanism) [53], and deglycosylation with subsequent backbone cleavage [54–56]. Oligonucleotide end modification has also been achieved by DNA 5′-phosphorylation (kinase activity) [57] and adenylylation (capping) [58] followed by ligation [59].…”

Characterization of deoxyribozymes with site-specific oxidative cleavage activity against DNA obtained by in vitro selection

“…Engineering deoxyribozymes that selectively cleave DNA is expected to be very challenging. The Silverman 13 and Breaker 14 groups conducted direct evolution experiments that have given rise to selective and rapid DNA-hydrolyzing deoxyribozymes. DNA cleavage by oxidative mechanisms has also been well-studied for its potential role in the pathophysiology of inflammation, cancer, and degenerative diseases.…”

Single‐Molecule Visualization of the Activity of a Zn2+‐Dependent DNAzyme

“…[4c, 12] High-speed atomic force microscopy (AFM) measurements were recently implemented to probe DNAn anostructures and their reactivity at the single-molecule level. [18] We connected the DNAzyme to one supported dsDNAm olecule and the substrate to another, and subsequently incorporated them into the DNAorigami frame by using the four single-stranded DNAt ethers as linkers (Figure 1b). [17] Because the target DNAs trands are directly attached to the nanoscaffold, the DNAs tructural changes can be controlled in the nanospace by arranging the structures inside the DNAn anoscaffold.…”

“…Fort he observation of substrate cleavage by the DNAzyme,w e introduced the DNAzyme and substrate between two doublestranded DNAm olecules (dsDNAs) in aD NA frame ( Figure 1). [18] We connected the DNAzyme to one supported dsDNAm olecule and the substrate to another, and subsequently incorporated them into the DNAorigami frame by using the four single-stranded DNAt ethers as linkers (Figure 1b). [18] We connected the DNAzyme to one supported dsDNAm olecule and the substrate to another, and subsequently incorporated them into the DNAorigami frame by using the four single-stranded DNAt ethers as linkers (Figure 1b).…”