A Post-Labeling Approach for the Characterization and Quantification of RNA Modifications Based on Site-Directed Cleavage by DNAzymes

Publikationstyp:
Zeitschriftenaufsatz
Metadaten:
Autoren
  • Madeleine Meusburger
  • Martin Hengesbach
  • Mark Helm
DOI
10.1007/978-1-61779-018-8_16
eISSN
1940-6029
ISSN
1064-3745
Online publication date
2011
Paginierung
259 - 270
Datum der Veröffentlichung
2011
Status
Published
Herausgeber
Humana Press
Herausgeber URL
http://dx.doi.org/10.1007/978-1-61779-018-8_16
Datum der Datenerfassung
2023
Titel
A Post-Labeling Approach for the Characterization and Quantification of RNA Modifications Based on Site-Directed Cleavage by DNAzymes

Datenquelle: Crossref

Andere Metadatenquellen:
Abstract
Deoxyribozymes or DNAzymes are small DNA molecules with catalytic activity originating from in vitro selection experiments. Variants of the two most popular DNAzymes with RNase activity, the 10-23 DNAzyme and the 8-17 DNAzyme, promote efficient in vitro cleavage of the phosphodiester bond in at least 11 out of 16 possible dinucleotide permutations. Judicious choice of the sequences flanking the active core of the DNAzymes permits to direct cleavage activity with high sequence specificity. Here, the harnessing of these features for the analysis of RNA nucleotide modifications by a post-labeling approach is described in detail. DNAzymes are designed such that RNase cleavage is directed precisely to the 5' end of the nucleotide to be analyzed. Iterative complex formation of DNAzyme and RNA substrate and subsequent cleavage are performed by temperature cycling. The DNAzyme activity liberates the analyte nucleotide on the very 5'-end of an RNA fragment, whose hydroxyl group can be conveniently phosphorylated with (32)P. The labeled RNA is digested to mononucleotides, and analyzed by thin layer chromatography.
Addresses
  • Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany.
Autoren
  • Madeleine Meusburger
  • Martin Hengesbach
  • Mark Helm
DOI
10.1007/978-1-61779-018-8_16
eISSN
1940-6029
Externe Identifier
  • PubMed Identifier: 21370054
Open access
false
ISSN
1064-3745
Zeitschrift
Methods in molecular biology (Clifton, N.J.)
Schlüsselwörter
  • Humans
  • DNA, Catalytic
  • Nucleotides
  • RNA
  • Chromatography, Thin Layer
  • Base Sequence
Sprache
eng
Medium
Print
Paginierung
259 - 270
Datum der Veröffentlichung
2011
Status
Published
Datum der Datenerfassung
2011
Titel
A post-labeling approach for the characterization and quantification of RNA modifications based on site-directed cleavage by DNAzymes.
Sub types
  • Research Support, Non-U.S. Gov't
  • Journal Article
Ausgabe der Zeitschrift
718

Datenquelle: Europe PubMed Central

Abstract
Deoxyribozymes or DNAzymes are small DNA molecules with catalytic activity originating from in vitro selection experiments. Variants of the two most popular DNAzymes with RNase activity, the 10-23 DNAzyme and the 8-17 DNAzyme, promote efficient in vitro cleavage of the phosphodiester bond in at least 11 out of 16 possible dinucleotide permutations. Judicious choice of the sequences flanking the active core of the DNAzymes permits to direct cleavage activity with high sequence specificity. Here, the harnessing of these features for the analysis of RNA nucleotide modifications by a post-labeling approach is described in detail. DNAzymes are designed such that RNase cleavage is directed precisely to the 5' end of the nucleotide to be analyzed. Iterative complex formation of DNAzyme and RNA substrate and subsequent cleavage are performed by temperature cycling. The DNAzyme activity liberates the analyte nucleotide on the very 5'-end of an RNA fragment, whose hydroxyl group can be conveniently phosphorylated with (32)P. The labeled RNA is digested to mononucleotides, and analyzed by thin layer chromatography.
Autoren
  • Madeleine Meusburger
  • Martin Hengesbach
  • Mark Helm
Autoren-URL
https://www.ncbi.nlm.nih.gov/pubmed/21370054
DOI
10.1007/978-1-61779-018-8_16
eISSN
1940-6029
Zeitschrift
Methods Mol Biol
Schlüsselwörter
  • Base Sequence
  • Chromatography, Thin Layer
  • DNA, Catalytic
  • Humans
  • Nucleotides
  • RNA
Sprache
eng
Country
United States
Paginierung
259 - 270
Datum der Veröffentlichung
2011
Status
Published
Datum, an dem der Datensatz öffentlich gemacht wurde
2011
Titel
A post-labeling approach for the characterization and quantification of RNA modifications based on site-directed cleavage by DNAzymes.
Sub types
  • Journal Article
  • Research Support, Non-U.S. Gov't
Ausgabe der Zeitschrift
718

Datenquelle: PubMed

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