Detection of RNA Hydrolysis with Binase by Acridine Orange Fluorescence

Abstract

Fluorescence spectroscopy was used to study the specific hydrolysis of ribosomal RNA by binase in aqueous buffer solutions. Acridine orange was used for RNA staining. When the dye was bound to RNA, its fluorescence intensity increased by two times due to the formation of a stable complex. During RNA hydrolysis by binase for 1–20 min, this complex was cleaved, which was accompanied by a nearly twofold decrease in fluorescence. The degree of polarization of the dye fluorescence during hydrolysis was reduced by a factor of 5.5. The hydrolysis reaction was slower at a pH of 5.0–6.0 than at a pH of 7.0–8.0, but it proceeded almost until the end. Hydrolysis was slowed with an increase in the ionic strength of the buffer and was suppressed by magnesium ions. The studied reaction can be further used as a convenient, selective, fluorometric method for the detection of single-stranded ribosomal RNA and the study of their properties.