Fluorescence postlabeling assay of RNA modification.
A new approach has been developed to assay RNA modification by combining enzymatic digestion of RNA to nucleoside monophosphate and fluorescence postlabeling. Nuclease P1 digestion of modified RNA affords both normal and modified nucleotides. The nucleotides are labeled in-situ with dansyl chloride via phosphoramidate derivatives with ethylenediamine following a procedure initially designed to synthesize fluorescence labeled deoxynucleotides. The postlabeled nucleotides are analyzed by HPLC using a fluorescence detector. Fluorescence postlabeling assay of 7-methylGmp in RNA exposed to dimethyl sulfate has validated the technique. In the presence of excess normal nucleotides, the limit of detection (LOD) lies between normal to modified nucleotide ratio of 10(3) to 10(4). HPLC enrichment of the modified nucleotide from the normal nucleotides prior to labeling enhances the LOD by two orders of magnitude.
Jain
R
R
Department of Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263.
Isac
T V
TV
Sharma
M
M
D002851:Chromatography, High Pressure Liquid; D008745:Methylation; D012313:RNA / Q000737:chemistry*; D012314:RNA Cap Analogs / Q000737:chemistry*; D012260:Ribonucleases / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D012314:RNA Cap Analogs; C011925:7-methylguanosine-5'-monophosphate; D012313:RNA; D012260:Ribonucleases