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Visualizing the dynamics of viral replication in living cells via Tat peptide delivery of nuclease-resistant molecular beacons

In this study, we describe the use of nuclease-resistant molecular beacons (MBs) for the real-time detection of coxsackievirus B6 replication in living Buffalo green monkey kidney (BGMK) cells via Tat peptide delivery. A nuclease-resistant MB containing 2′-O-methyl RNA bases with phosphorothioate internucleotide linkages was designed to specifically target an 18-bp 5′ noncoding region of the viral genome. For intracellular delivery, a cell-penetrating Tat peptide was conjugated to the MB by using a thiol–maleimide linkage. Presence of the Tat peptide enabled nearly 100% intracellular delivery within 15 min. When the conjugate was introduced into BGMK cell monolayers infected with coxsackievirus B6, a discernible fluorescence was observed at 30 min after infection, and as few as 1 infectious viral particle could be detected within 2 h. The stability and the intracellular delivery properties of the modified MBs enabled real-time monitoring of the cell-to-cell spreading of viral infection. These results suggest that the Tat-modified, nuclease-resistant MBs may be powerful tools for improving our understanding of the dynamic behavior of viral replication and for therapeutic studies of antiviral treatments.