Use of DNAzymes for site-specific analysis of ribonucleotide modifications

Abstract

Post-transcriptional ribonucleotide modifications are widespread and abundant processes that have not been analyzed adequately due to the lack of appropriate detection methods. Here, two methods for the analysis of modified nucleotides in RNA are presented that are based on the quantitative and site-specific DNAzyme-mediated cleavage of the target RNA at or near the site of modification. Quantitative RNA cleavage is achieved by cycling the DNAzyme and its RNA substrate through repeated periods of heating and cooling. In a first approach, DNAzyme-directed cleavage directly 5′ of the residue in question allows radioactive labeling of the newly freed 5′-OH. After complete enzymatic hydrolysis, the modification status can be assessed by two-dimensional thin layer chromatography. In a second approach, oligoribonucleotide fragments comprising the modification site are excised from the full-length RNA in an endonucleolytic fashion, using a tandem DNAzyme. The excised fragment is isolated by electrophoresis and submitted to further conventional analysis. These results establish DNAzymes as valuable tools for the site-specific and highly sensitive detection of ribonucleotide modifications.