Grouping of ferritin and gold nanoparticles conjugated to pRNA of the phage phi29 DNA-Packaging motor

The bacteriophage phi29 DNA-packaging motor, which translocates and compresses the DNA genome of the phage into its procapsid during virion assembly, involves an essential ring formed by the packaging RNA (pRNA). We attached electron-dense nanoparticles to pRNA by hybridizing a DNA oligonucleotide with a biotin or thiol modification to a 3'-extension of core pRNA, and by coupling streptavidin and biotinylated ferritin, or 5 nm and 10 nm colloidal gold particles, to these modifications. The pRNA conjugates bound to RNA-free phi29 procapsids, and such nanoparticle-bearing procapsids were isolated by ultracentrifugation and analyzed. Electron microscopy showed that ferritin and gold particles were specifically attached to the side of the phi29 procapsid harboring the connector, which is the pRNA binding site. The pRNA-ferritin conjugates bound to procapsids with the same efficiency as pRNA which lacked the high molecular mass label. However, full DNA packaging efficiency in an in vitro phage assembly assay was only reached after the label of such isolated procapsid-pRNA complexes had been released with RNase H. The results provide approaches to assembled ferritin or gold-containing nano-complexes via pRNA mediated assembly.