Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA

Ying Xiao, Rebecca J. Wehrmann, Nora A. Ibrahim, Scott K Silverman

Research output: Contribution to journalArticle

Abstract

We recently reported that a DNA catalyst (deoxyribozyme) can site-specifically hydrolyze DNA on the minutes time scale. Sequence specificity is provided by Watson-Crick base pairing between the DNA substrate and two oligonucleotide binding arms that flank the 40-nt catalytic region of the deoxyribozyme. The DNA catalyst from our recent in vitro selection effort, 10MD5, can cleave a single-stranded DNA substrate sequence with the aid of Zn 2+ and Mn 2+ cofactors, as long as the substrate cleavage site encompasses the four particular nucleotides ATG^T. Thus, 10MD5 can cleave only 1 out of every 256 (4 4) arbitrarily chosen DNA sites, which is rather poor substrate sequence tolerance. In this study, we demonstrated substantially broader generality of deoxyribozymes for site-specific DNA hydrolysis. New selection experiments were performed, revealing the optimality of presenting only one or two unpaired DNA substrate nucleotides to the N 40 DNA catalytic region. Comprehensive selections were then performed, including in some cases a key selection pressure to cleave the substrate at a predetermined site. These efforts led to identification of numerous new DNA-hydrolyzing deoxyribozymes, many of which require merely two particular nucleotide identities at the cleavage site (e.g. T^G), while retaining Watson-Crick sequence generality beyond those nucleotides along with useful cleavage rates. These findings establish experimentally that broadly sequence-tolerant and site-specific deoxyribozymes are readily identified for hydrolysis of single-stranded DNA.

Original languageEnglish (US)
Pages (from-to)1778-1786
Number of pages9
JournalNucleic acids research
Volume40
Issue number4
DOIs
StatePublished - Feb 1 2012

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Single-Stranded DNA
Hydrolysis
Catalytic DNA
DNA
Nucleotides
Catalytic Domain
Oligonucleotides
Base Pairing
Pressure

ASJC Scopus subject areas

  • Genetics

Cite this

Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA. / Xiao, Ying; Wehrmann, Rebecca J.; Ibrahim, Nora A.; Silverman, Scott K.

In: Nucleic acids research, Vol. 40, No. 4, 01.02.2012, p. 1778-1786.

Research output: Contribution to journalArticle

Xiao, Ying ; Wehrmann, Rebecca J. ; Ibrahim, Nora A. ; Silverman, Scott K. / Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA. In: Nucleic acids research. 2012 ; Vol. 40, No. 4. pp. 1778-1786.
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