Construction and 3-D Computer Modeling of Connector Arrays with Tetragonal to Decagonal Transition Induced by pRNA of phi29 DNA-Packaging Motor

Abstract

The bottom-up assembly of patterned arrays is an exciting and important area in current nanotechnology. Arrays can be engineered to serve as components in chips for a virtually inexhaustible list of applications ranging from disease diagnosis to ultrahigh-density data storage. In attempting to achieve this goal, a number of methods to facilitate array design and production have been developed. Cloning and expression of the gene coding for the connector of the bacterial virus phi29 DNA-packaging motor, overproduction of the gene products, and the in vitro construction of large-scale carpet-like arrays composed of connector are described in this report. The stability of the arrays under various conditions, including varied pH, temperature and ionic strength, was tested. The addition of packaging RNA (pRNA) into the array caused a dramatic shift in array structure, and resulted in the conversion of tetragonal arrays into larger decagonal structures comprised of both protein and RNA. RNase digestion confirmed that the conformational shift was caused by pRNA, and that RNA was present in the decagons. As has been demonstrated in biomotors, conformational shift of motor components can generate force for motor motion. The conformational shift reported here can be utilized as a potential force-generating mechanism for the construction of nanomachines. Three-dimensional computer models of the constructed arrays were also produced using a variety of connector building blocks with or without the N- or C-terminal sequence, which is absent from the current published crystal structures. Both the connector array and the decagon are ideal candidates to be used as templates to build patterned suprastructures in nanotechnology.

Interaction of gp16 with pRNA and DNA for Genome Packaging by the Motor of Bacterial Virus phi29

“…pRNA dimers and trimers, and the rest of the motor as a whole, have been shown to have significant potential to be used as parts in biological nanotechnology. [7][8][9][97][98][99] …”

“…2 Factors involved in the DNA-packaging motor and its components have the potential to be used as building blocks in nanotechnology. [7][8][9] This essential viral replication step has been investigated in the quest for model targets for the inhibition of viral replication 10,11 and for the development of drugs against cytomegalovirus (CMV). 12 An examination of the well-studied dsDNA viruses reveals that the motor involves a dodecameric protein connector [13][14][15][16] and two non-structural components with certain characteristics typical of an ATPase.…”

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

“…4A). The pRNA ring showed an outer contour diameter of ∼30 nm formed through the looploop interactions, with a center ring of ∼10 nm in diameter to sheath on the connector that has an outer contour diameter of 9.4 nm for its narrower end and 13.8 nm for its wider end (Guo et al 2005b). A comparison of the AFM images with the pRNA hexamer model showed a strong correlation in shape and size, including sixfold rotational axes and protrusions every 60°(these correspond to helix H1, essential for contacting the ATPase).…”

“…The wide end is embedded inside the capsid, and the narrow end is exposed for interaction with the pRNA (Jimenez et al 1986;Simpson et al 2000;Guo et al 2005b). The interaction between pRNA and the connector was demonstrated by crosslinking of the two (Garver and Guo 1997).…”

Binding of pRNA to the N-terminal 14 amino acids of connector protein of bacteriophage phi29

“…Owing to the structural flexibility of both the N- and C-terminal regions, the crystal structure of 13 amino acids at the N-terminus and 24 amino acids at the C-terminus were not solved and were missing from PDB file 1H5W (34), while 10 amino acids at the N-terminus and 24 amino acids at the C-terminus were missing from PDB file 1FOU (35). From structural analysis, it is evident that the N-terminus protrudes from the narrow end of the connector and the C-terminus protrudes from the wider end of the connector (Figure 1) (34–36). The C-terminus is buried within the procapsid and the N-terminus is exposed to the solvent.…”