Nucleic Acids Research, 1984, Vol. 12, No. 2 989-1002
© 1984
CHEMISTRY |
Attachment of reporter groups to specific, selected cytidine residues in RNA using a bisulfitecatalyzed transamination reaction
Johns Hopkins University Baltimore, MD 21218, USA
Received October 18, 1983. Revised December 13, 1983. Accepted December 13, 1983.
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°C with ethylene diamine (pK1= 7.6) and bisulfite. This derivative reacts quantitatively with N-hydroxysuccinimide esters; the linkage of a fluorescent dye, nitrohenzofurazan, to cytidines 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 rihosomal RNA have been modified by this technique.