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Fluorescent microscopy experiments show that when 2'-O-methyl-modified molecular beacons (MBs) are introduced into NIH/3T3 cells, they elicit a nonspecific signal in the nucleus. This false-positive signal can be avoided by conjugating MBs to macro- molecules (e.g. NeutrAvidin) that prevent nuclear sequestration, but the presence of a macromolecule makes efficient cytosolic delivery of these probes challenging. In this study, we explored various methods including TAT peptide, Streptolysin O and microporation for delivering NeutrAvidin-conjugates into the cytosol of living cells. Surprisingly, all of these strategies led to entrapment of the conjugates within lysosomes within 24h. When the conjugates were pegylated, to help prevent intracellular recog- nition, only microporation led to a uniform cytosolic distribution. Microporation also yielded a trans- fection efficiency of 93% and an average viability of 86%. When cells microporated with MB-NeutrAvidin conjugates were examined via flow cytometry, the signal-to-background was found to be more than 3 times higher and the sensitivity nearly five times higher than unconjugated MBs. Overall, the present study introduces an improved methodology for the high-throughput detection of RNA at the single cell level.

Efficient cytosolic delivery of molecular beacon conjugates and flow cytometric analysis of target RNA