SPECIFIC DETECTION OF RNA MOLECULES BY FLUORESCENT IN-SITU HYBRIDIZATION IN LIVING CELLS

The antisense therapeutic strategy makes the assumption that sequence- specific hybridization of an oligonucleotide to its target can take pl ace in living cells. The present work provides a new method for the de tection of intracellular RNA molecules using in situ hybridization on living cells. The first step consisted in designing nonperturbant cond itions for cell permeabilization using streptolysin O. In a second ste p, intracellular hybridization specificity was evaluated by incorporat ing various types of fluorescently labeled nucleic acid probes (plasmi ds, oligonucleotides). Due to its high expression level, the 28S ribos omal RNA was retained as a model. Results showed that: (1) no signific ant cell death was observed after permeabilization; (2) on living cell s, 28S RNA specific probes provided bright nucleoli and low cytoplasmi c signal; (3) control probes did not lead to significant fluorescent s taining; and (4) comparison of signals obtained on living and fixed ce lls showed a colocalization of observed fluorescence. These results in dicate the feasibility of specific hybridization of labeled nucleic ac id probes under living conditions, after a simple and efficient permea bilization step. This new detection method is of interest for investig ating the dynamics of distribution of various gene products in living cells, under normal or pathological conditions.