In vitroselection of DNA-cleaving deoxyribozyme with site-specific thymidine excision activity

Abstract

Single-nucleotide polymorphisms, either inherited or due to spontaneous DNA damage, are associated with numerous diseases. Developing tools for site-specific nucleotide modification may one day provide a way to alter disease polymorphisms. Here, we describe the in vitro selection and characterization of a new deoxyribozyme called F-8, which catalyzes nucleotide excision specifically at thymidine. Cleavage by F-8 generates 3′- and 5′-phosphate ends recognized by DNA modifying enzymes, which repair the targeted deoxyribonucleotide while maintaining the integrity of the rest of the sequence. These results illustrate the potential of DNAzymes as tools for DNA manipulation.

Theranostic DNAzymes

“…The current discussion has been focused on RNA-cleaving DNAzymes, but other systems can be easily envisioned. For example, DNA-cleaving DNAzymes were also implemented for metal sensing 161-163, and their therapeutic potential was demonstrated for DNA excision repair 164. In addition, aptazymes might be developed to respond to glucose.…”

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

“…In sharp contrast, the Cu 2+ , Mn 2+ -dependent deoxyribozyme F-8 we reported previously oxidatively catalyzed nucleotide excision, leading to 3′-and 5′-phosphate ends. 16 Having characterized the catalytic activity of the deoxyribozymes, we next analyzed the chemical requirements, substrate sequence tolerance and detailed secondary structures for their activities. A-2 was severely inhibited in the absence of Mn 2+ or Cu 2+ .…”

“…12 Recently, we reported a new deoxyribozyme F-8 which could oxidatively catalyze nucleotide excision specifically at thymidine in a single-stranded DNA substrate with Cu 2+ and Mn 2+ as cofactors. 16 As part of our overall efforts to investigate the catalytic abilities of DNA, in the present study, we would like to identify more additional site-specific DNA-oxidating deoxyribozymes using the two typical metal ions. Via new experiments, here we found a new class of Cu 2+ and Mn 2+ -dependent DNA-cleaving deoxyribozymes named A-2 and A-3.…”

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].…”