Figure 4.

Thin-layer chromatographic separation of the hydrolysis products of the target RNA. The standard 29-nt long target RNA contains a single unique UG dinucleotide. The U residue in this dinucleotide is the expected site of methylation and corresponds to position U52 in 16S rRNA (A). Guide and target RNAs were mixed in a 20-μl reaction in the standard methylation assay using [methyl-³H]SAM and incubated at 70°C for 1 h. The RNA was extracted, digested with 0.01 unit of P1 nuclease (Roche Diagnostics) for 12 h at 37°C, and products were mixed with 5 nmol each of pAm, pCm, pGm, pUm, and pUmG standards before the two-dimensional TLC separation (28). Unlabeled standards were detected by UV shading and radioactivity was detected by ³H-imaging (B). The spots corresponding to the UV-detectable standards were excised from the plate and subjected to scintillation counting (see text). Omission of any of the RNA or protein components from the reaction mixture results in background levels of incorporation of radioactivity into acid-insoluble material (see Fig. 2).