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. 2008 Jan;14(1):180-7.
doi: 10.1261/rna.742708. Epub 2007 Nov 12.

Use of DNAzymes for site-specific analysis of ribonucleotide modifications

Martin Hengesbach  1 , Madeleine Meusburger, Frank Lyko, Mark Helm
Affiliations

Use of DNAzymes for site-specific analysis of ribonucleotide modifications

Martin Hengesbach et al. RNA. 2008 Jan.

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.

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Figures

FIGURE 1.
FIGURE 1.
Post-labeling after site-specific DNAzyme mediated cleavage. (A) Overview of the experimental procedure, showing annealing of the DNAzyme to the target RNA, separation by PAGE of the fragments resulting from cleavage, and TLC analysis after digestion to mononucleotides. The middle panel shows a phosphorimager scan of a denaturing PAGE of fragmented tRNALys after temperature cycling for the indicated number of times. C denotes a control lane with RNA incubated without DNAzyme. Lanes labeled “unmodified” contain untreated tRNALys transcript, lanes labeled “Pus1p-modified” contain tRNALys transcript treated with Pus1p enzyme prior to analysis. The downstream fragment resulting from DNAzyme cleavage, indicated by “fragment of interest” in the figure, was excised for further analysis by 5′ labeling with 32P, digestion to mononucleotides, and thin layer chromatography. The RNA in the gel is internally labeled with [α-32P]-UTP to facilitate cleavage detection. Note that the actual analysis is performed on RNA, which is nonradioactive until the point of post-labeling. (Lower left) TLC of a series of calibration samples with known U/Ψ composition at position 27 after application of the analytic procedure. The spots of U and Ψ are indicated. (Lower right) Application to the analysis of tRNALys incubated with Pus1p. Times of incubation with Pus1p are indicated in minutes; C designates a nonmodified control. (B) DNAzyme design for targeting residue Ψ/U 27 in human mitochondrial tRNALys. The target tRNA is shown in its unusual extended hairpin form (Helm et al. 1998). The hybridization sequences of DNAzyme I are indicated by a bold line. The loop in the bold line indicates the catalytic center near the targeted cleavage site. Pseudouridine synthase 1 and its respective target site for post-transcriptional modification of uridine to pseudouridine (Ψ) are indicated. (C) Cleavage efficiency is affected by modifications. Target cleavage yields of unmodified tRNA (solid squares) and a tRNA pre-incubated with Pus1p (open triangles) are compared as a function of the number of temperature cycles. (D) Validation of detection efficiency. The composition of a mixture of tRNAs containing defined ratios of Ψ/U 27 is plotted versus the ratio detected after site-specific labeling.
FIGURE 2.
FIGURE 2.
Tandem DNAzyme-mediated excision of target oligoribonucleotides for modification analysis. (A) Design of a tandem DNAzyme for double cleavage of RNA. Scissors indicate cleavage sites on the RNA. The catalytic centers of the DNAzyme are displayed as loops. (B, top) Secondary structure of 3′-truncated tRNAAsp. The hybridization sequences of DNAzymes II (bold line) and III (dotted line) are indicated. Loops indicate the catalytic centers near the targeted cleavage site. Methyltransferases Dnmt2 and Trm4 and their respective target sites for post-transcriptional modification to 5-methylcytosine are indicated. (Bottom) RNA fragments resulting from the combined action of DNAzymes II and III. Scissors indicate cleavage sites on the RNA. (C) PhosphorImager scan of fragments generated by DNAzyme-directed cleavage. Lanes designated II, III, and II+III show incubations in the presence of the respective DNAzymes, and C designates the control incubation without DNAzyme. The size of the RNA fragments is indicated on the right. Arrows indicate fragments that have been isolated from a reaction after cycling to near-complete hydrolysis. (D) 2D-TLC of mononucleotide mixtures resulting from the excised 9-mer and 18-mer fragments of [α-32P]-CTP labeled RNA after incubation with Dnmt2 and Trm4, respectively. pC and pm5C identify spots corresponding to the 5′-phosphates of cytosine and 5-methylcytosine, p stands for free phosphate, and pGp for 5′-pG-cyclic-2′-3′-[32P]-p diphosphate, generated from the 3′ end of the 9-mer by P1-digestion. Dimensions of chromatography in solvents A and B are indicated by arrows.
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