During assembly, bacteriophage phi29 utilizes a motor to insert genomic DNA into a preformed protein shell called the procapsid. The motor contains one twelve-subunit head-tail connector gp10, a packaging RNA (pRNA) ring, and an ATPase protein, gp16, with unknown stoichiometry. It was proposed that the symmetry mismatch between the five-fold procapsid shell and twelve-fold connector (or the hexameric pRNA ring) produces a force to drive a rotation motor to translocate and compress DNA. There were discrepancies regarding the location of the foothold for the pRNA and whether the pRNA ring is a hexamer or a pentamer. To elucidate the mechanism of phi29 DNA packaging, both purified connector and purified procapsid were used for binding studies with in vitro synthesized pRNA. Removal of the N-terminal fourteen amino acids of the gp10 protein by proteolytic cleavage resulted in undetectable binding of pRNA to either the free connector or the procapsid. It is therefore concluded that pRNA bound to the 12-fold symmetrical connector to form a pRNA-connector complex and that the foothold for pRNA is the connector but not the capsid protein. Further work on the purified connectors showed that diverse RNA or DNA binds to the connector nonspecifically and with similar ability. However a combined pRNA ring-forming group was very specific for motor binding. It is generally believed that the specificity in RNA/protein interaction relies on molecular contact through a surface charge, or 3D structure matching. Here we report a new mechanism suggesting that the specificity and affinity of RNA/protein interaction relies on RNA static ring formation. pRNA did not form a ring prior to motor binding. Only those RNAs that formed a static ring, via the interlocking loops, stayed on the motor. Single interlocking loop interruption resulted in pRNA detachment. Extension or reduction of the ring circumference failed in motor-binding. This new mechanism was tested by redesigning two artificial RNAs that formed hexamer and packaged DNA. The results also confirmed that the stoichiometry of pRNA on the motor was a common multiple of two and three, thus, a hexamer.
To build a synthetic nanomotor which mimics the functions of phi29 DNA packaging motor, connector protein was reengineered, and a thin lipid monolayer was used to direct the assembly of massive sheets of single layer patterned arrays of the reengineered motor dodecamer. Uniform, clean and highly-ordered arrays were constructed as shown by both transmission electron microscopy and atomic force microscopy imaging. The reengineered connector protein was also found to have the ability of forming a 20 x 30 nm ellipsoid nanoparticle containing 84 subunits or 7 connectors. The formation of this structure was mediated and stabilized by N-terminal peptide extensions. Reversal of the 84-subunit ellipsoid nanoparticle to its dodecamer subunit was controlled by the cleavage of the extended N-terminal peptide with a protease. The 84 outward-oriented C-termini were conjugated with a streptavidin binding peptide which can be used for the incorporation of markers. This further extends the application of this nanoparticle to pathogen detection and disease diagnosis by signal enhancement.