Conformation of RNA in situ as studied by acridine orange staining and automated cytofluorometry

Abstract

Secondary structure of RNA was studied in human leukemic SK-L7 cells by their staining with acridine orange (AO) and fluorescence measurements in a flow-through cytofluorimeter. Parallel measurements of RNAse-treated cells made it possible to distinguish and to measure separately those components of total cell fluorescence that are due to AO interaction with RNA. Like DNA, double helical and single-stranded RNA in situ stain differentially with AO. Hence, the extent of RNA in double helical conformation, interacting with AO by intercalation, can be measured. In 10 −3M phosphate buffer, RNA in situ denatures at 40–60 °C. Mg ions stabilize RNA against thermal denaturation; EDTA markedly decreases stability of RNA in double helical conformation. A slow, progressive denaturation of RNA in situ is seen as a result of cell exposure to AO at low ionic strength at room temperature. It is presumed that the changes described reflect mostly alterations of secondary structure of rRNA. Results of our in situ studies conform with data of others on rRNA denaturation in intact isolated ribosomes. This suggests that the ribosome anchoring in cytoplasm is not associated with double helical rRNA regions. Cell staining with AO under conditions of full RNA denaturation and in the absence of DNA denaturation offers differential, simultaneous staining of RNA and DNA. This approach in cell staining provides a new parameter for automated cell classification based on both quantity and conformation of RNA in situ.