Comparing Agent‐Based Delivery of DNA and PNA Forced Intercalation (FIT) Probes for Multicolor mRNA Imaging

Fluorogenic oligonucleotide probes allow mRNA imaging in living cells. A key challenge is the cellular delivery of probes. Most delivery agents, such as cell‐penetrating peptides (CPPs) and pore‐forming proteins, require interactions with the membrane. Charges play an important role. To explore the influence of charge on fluorogenic properties and delivery efficiency, we compared peptide nucleic acid (PNA)‐ with DNA‐based forced intercalation (FIT) probes. Perhaps counterintuitively, fluorescence signaling by charged DNA FIT probes proved tolerant to CPP conjugation, whereas CPP–FIT PNA conjugates were affected. Live‐cell imaging was performed with a genetically engineered HEK293 cell line to allow the inducible expression of a specific mRNA target. Blob‐like features and high background were recurring nuisances of the tested CPP and lipid conjugates. By contrast, delivery by streptolysin‐O provided high enhancements of the fluorescence of the FIT probe upon target induction. Notably, DNA‐based FIT probes were brighter and more responsive than PNA‐based FIT probes. Optimized conditions enabled live‐cell multicolor imaging of three different mRNA target sequences.