Mapping of Complete Set of Ribose and Base Modifications of Yeast rRNA by RP-HPLC and Mung Bean Nuclease Assay
Fig 2
Mung bean nuclease protection assay.
A) Schematic illustration of MBN protection assay used in the present study for the analysis of chemical modifications of 18S rRNA, and B) 25S rRNA. 45 distinct fragments from 18S, and 68 separate fragments from 25S rRNA were isolated for mapping of their chemical constituents. C) Graphical illustration for the use of tiling set of overlapping fragments used in the present study to map the modified residues to a single nucleotide resolution. The fragments protected from MBN were isolated from the debris by 7 M Urea 13% PAGE gel. D) Representative gel for the MBN assay showing an intact protected fragment retrieved after the MBN digestion. The synthetic antisense oligonucleotide used for the protection is used as a marker (50 nts). All fragments isolated from 18S and 25S rRNA were extracted from the gel and digested to nucleosides for their composition analysis.