Assembly mechanism of the sixty-subunit nanoparticles via interaction of RNA with the reengineered protein connector of phi29 DNA-packaging motor

Abstract

Bacterial virus phi29 genomic DNA is packaged into a procapsid shell with the aid of a motor containing a 12-subunit connector channel and a hexameric pRNA (packaging RNA) ring. The wide end, or the C-terminus, of the cone-shaped connector is embedded within the procapsid shell, whereas the narrow end, or N-terminus, extends outside of the procapsid, providing a binding location for pRNA. Recently, we have reported the mechanism of in vivo assembly of an ellipsoid nanoparticle with seven connectors through an interaction among a peptide tag. Here we report the formation of a similar nanoparticle in vitro via the addition of DNA or RNA oligos to connector proteins. Free connectors guided by one or two copies of oligonucleotides were assembled into a rosette structure containing 60 subunits of reengineered proteins. The number of oligonucleotides within the particle is length-dependent but sequence-independent. Reversible shifting between the 12- and 60-subunit nanoparticles (between individual connectors and rosette structures, respectively) was demonstrated by the alternative addition of oligonucleotides and the treatment of ribonuclease, suggesting a potential application as a switch or regulator in nanobiotechnology. This advancement allows for a simple method to produce multivalent nanoparticles that contain five 12-unit nanoparticles with defined structure and stoichiometry. That is, it will be possible to assemble nanoparticles in vitro with the combination of 60 assortments of ligands, tags, therapeutic drugs, and diagnostic moieties for multivalent delivery or enhancement of signal detection in nanotechnological and nanomedicinal applications.

Figure 1

Schematic illustration of procapsid−pRNA interaction.

Figure 2

Gel shift assay to compare the binding capacity between different RNAs and the connector. (A) Connector with dimer Cd′−Dc′; (B) connector with monomer Cd′; (C) connector with tRNA. The triangle indicates a 2-fold increase in RNA concentration. The complex was separated by 0.8% agarose gel. The gel was stained by ethidium bromide to track the RNA bands first (left), and then the same gel was stained by Coomassie brilliant blue to detect protein bands (right). Connector/tRNA (D) and connector/Aa′ (E) were analyzed to determine the binding constant. Lane 1 indicates RNA only. Triangle above lanes 2−10 indicates a 2-fold increase in connector concentration.

Figure 3

5−20% sucrose gradient sedimentation to detect the binding efficiency of pRNA to connector. Connector with pRNA Aa′ (closed diamond), Cd′ (open square), Cd′−Dc′ (open triangle), and tRNA (dot) were analyzed by 5−20% sucrose gradient sedimentation. All RNAs were labeled by [³H]. [³H]-Aa′ without connector (closed triangle) was used as a control.

Figure 4

Comparison of connector binding ability to DNA and RNA of the same length. A fixed amount of connector was mixed with 55 nt ssDNA, dsDNA, and ssRNA at different ratios. The mixtures were separated by 0.8% agarose gel. The gel was first stained with ethidium bromide to detect nucleic acids (A) and then stained by Coomassie brilliant blue to detect the proteins (B).

Figure 5

Negative stained electron micrograph images of connector and rosette. (A) Mixture of connector and pRNA; (B) purified connector−pRNA complex. Bar = 100 nm.

Figure 6

(A) Connector binding to nucleic acids is not purine- or pyrimidine-dependent. Constant amount of connector was mixed with various amounts of 20 nt poly-d(purine) and poly-d(pyrimidine), and the mixtures were analyzed by 0.8% agarose gel shift assays, followed by Coomassie brilliant blue staining. (B) Rosette formation by connector−nucleic acid interaction is sequence-length-dependent. Different lengths of DNA oligos were mixed with connector and analyzed by agarose gel electrophoresis. The connector protein itself appears as a smeared band in the gel, but the band pattern becomes more defined with increasing length of ssDNA.

Figure 7

Single molecule photobleaching analysis on rosette particle formed by connector−RNA interaction. (A−F) Histogram showing the frequency of photobleaching steps of Cy3-RNA in connector−RNA complex excited by a 532 nm laser beam. Each step in photobleaching represents the presence of one Cy3-RNA molecule. In the complex of connector−pRNA (Cd′−Dc′), the Cy3 molecule was only attached to the pRNA Cd′.

Figure 9

Illustration of mechanism in rosette formation. (A) Five connectors arranged into a pentagonal structure via side-to-side interactions with its external hydrophilic−hydrophobic−hydrophilic property. (B−D) Interaction of oligonucleotides with the narrow end of connectors containing “RKR” at the N-terminus of each gp10 subunit. Formation of rosette can be promoted by only one long oligo (DNA/RNA) (B), by short oligos to link the neighboring connectors (C), or by multiple overlapping oligos (D).

Figure 8

Reversible structural shift between the connector and the rosette as induced by nucleic acids and ribonuclease. Connector (lane 1) was transformed into a rosette by ssDNA (lane 2). After treating the connector−ssDNA complex by Mung Bean Nuclease (lane 3), ssDNA was degraded and the rosette reverted into free connector protein. The rosette was formed again when pRNA was added into the solution (lane 4). Rosette was dissociated into the connector when the connector−pRNA complex was treated by RNase A (lane 5). Lane 6, control of connector−pRNA complex only. Lanes 7−10, negative control without connector. The gel was first stained with ethidium bromide to detect nucleic acids (A) and then stained by Coomassie brilliant blue to detect the proteins (B).

Figure 10

(A−C) Genetic algorithm−Monte Carlo analysis of sedimentation velocity data: frictional ratio vs sedimentation coefficients for three different ratios of connector to RNA. The color gradient legend in each panel indicates the relative concentrations based on color. (D) Cartoons suggesting a mechanism of rosette formation. In the beginning, one RNA molecule interacts with one connector molecule, and then another connector joins the interaction. A stoichiometry of one RNA for each connector appears likely because a molar excess of RNA only increases the partial concentration of the free RNA (∼5 S).