Fluorescent labeling of plasmid DNA for gene delivery: Implications of dye hydrophobicity on labeling efficiencies and nanoparticle size

Covalent fluorescent labels are important tools for monitoring the in vitro and in vivo localization of plasmid DNA nanoparticles, but must meet several criteria including high DNA labeling efficiencies and minimal impact on nanoparticle size. We developed a novel fluorescent labeling strategy utilizing an aryl azide photolabel conjugated to a short cationic peptide to label plasmid DNA with Cyanine 5 and sulfo-Cyanine 5. Using a simple camera flash apparatus, photolabel-peptide-dyes can be conjugated to DNA in minutes with preservation of DNA structure and minimal dye photobleaching. The addition of two anionic sulfonates to the Cyanine 5 core greatly improved labeling efficiencies from ~13 to ~53% and mitigated PEGylated polyacridine peptide-DNA nanoparticle size increases over a range of labeling densities. Comparison of our sulfo-Cyanine 5 peptide label to the Mirus Bio Label IT-Cy5 kit revealed that while both did not affect nanoparticle sizes appreciably, labeling efficiencies with our conjugate were higher, possibly due to the higher positive charge density on the peptide linker. The results from this work provide important considerations for choosing fluorophore tags to track DNA nanoparticles.