Fluorescent labeling of fragments of high molecular weight RNA

Abstract

We describe a method to fluorescently label microgram quantities of high molecularweight RNA with acriflavine. The method involves hydrolyzing the RNA with HCl at pH 1.0 for 10 min to obtain segments of about 80 nucleotides. The 3′-terminal phosphate is removed from the ribose with alkaline phosphatase, and the terminal ribose is oxidized with periodate to form dialdehydes. Acriflavine is bound to the dialdehyde by the formation of a Schiff's base, and unbound acriflavine is removed by dialysis followed by chromatography on a Sephadex G-25 column eluted with phosphate buffered guanidine-HCl. Human 18 S rRNA bound 0.94 acriflavine molecules per 100 nucleotides and had a fluorescence excitation maximum at 460 nm and an emission maximum at 508 nm. If the hydrolysis step was omitted, this RNA bound only 0.12 acriflavine molecule per 100 nucleotides. Acriflavine-labeled high molecular weight yeast RNA showed a fluorescent intensity which was proportional to RNA concentration to a 1000-fold dilution.