Attachment of reporter groups to specific, selected cytidine residues in RNA using a bisulfite-catalyzed transamination reaction.
Bisulfite catalyzes transamination of cytidine at the N4 position; the suitability of this reaction for attaching reporter groups to selected cytidine residues in RNA molecules has been investigated. Poly(C) is nearly quantitatively converted to the poly (N4 aminoethyl-C) derivative after 3 hrs at 42 degrees C with ethylene diamine (pK1 = 7.6) and bisulfite. This derivative reacts quantitatively with N-hydroxysuccinimide esters; the linkage of a fluorescent dye, nitrobenzofurazan, to cytidine by this reaction is demonstrated. To direct the bisulfite reaction to selected cytidines within a large RNA molecule, the RNA is hybridized to complementary DNA containing a deletion. Only the cytidines in the single strand RNA loop (corresponding to the DNA deletion) are reactive. Two cytidines in the middle of a 340 base RNA fragment from 16S ribosomal RNA have been modified by this technique.
D000588:Amines; D000596:Amino Acids; D000975:Antioxidants; D001483:Base Sequence; D001572:Benzofurans; D055598:Chemical Phenomena; D002621:Chemistry; D003001:Cloning, Molecular; D003562:Cytidine; D005822:Genetic Vectors; D007202:Indicators and Reagents; D009690:Nucleic Acid Conformation; D011066:Poly C; D012313:RNA; D013447:Sulfites
D000588:Amines; D000596:Amino Acids; D000975:Antioxidants; D001572:Benzofurans; D007202:Indicators and Reagents; D013447:Sulfites; D011066:Poly C; C036334:2-nitrobenzofuran; D003562:Cytidine; D012313:RNA; C009279:sodium bisulfite