Single Molecule Sensitive Multivalent Polyethylene Glycol Probes for RNA Imaging

Abstract

The imaging of RNA in live cells presents a methodological challenge both in the mechanism by which the probes are delivered to the cell and in the sensitivity of the probe. Probes must be delivered to the correct cellular compartment and, once inside the cell, should bind to the target RNA rapidly and with enough sensitivity to detect small numbers of RNA molecules. Here, we report the characterization of a single-molecule sensitive, multivalent RNA imaging probe that utilizes an eight-armed poly(ethylene glycol) core. This probe allowed for the accurate imaging of the human respiratory syncytial virus (hRSV) genomic RNA when delivered into live cells using either reversible membrane permeabilization or TAT-peptide mediated membrane transduction. Neither reversible membrane permeabilization nor TAT mediated delivery proceeded through the endosomal pathway, and both delivery methods demonstrated fast binding kinetics of less than 10 min. Subcellular distributions of RNA were visualized in live cells, as well as RNA-protein colocalization, after fixation and immunostaining of the hRSV RNA binding protein N.