Bacterial virus phi29 DNA-packaging motor and its potential applications in gene therapy and nanotechnology

Abstract

A controllable, 30-nm imitating DNA-packaging motor was constructed. The motor is driven by six synthetic adenosine triphosphate (ATP)-binding RNA (packaging RNA [pRNA]) monomers, similar to the driving of a bolt with a hex nut. Conformational change and sequential action of the RNA with fivefold (viral capsid)/sixfold (pRNA hexamer) mismatch could ensure continuous rotation of the motor with ATP as energy. In the presence of ATP and magnesium, a 5-microm synthetic DNA was packaged using this motor. On average, one ATP was used to translocate two bases of DNA. The DNA-filled capsids were subsequently converted into up to 109 PFU/mL of infectious virus. The three-dimensional structures of pRNA monomer, dimer, and hexamer have been probed by photoaffinity crosslinking, chemical modification interference, cryo-atomic force microscopy, and computer modeling. The pRNA's size and shape can be controlled and manipulated at will to form stable dimers and trimers. Cryo-atomic force microscopy revealed that monomers, dimers, and trimers displayed a checkmark outline, elongated shape, and triangular structure, respectively. The motor can be turned off by gamma-S-ATP or EDTA and turned on again with the addition of ATP or magnesium, respectively. The formation of ordered structural arrays of the motor complex and its components, the retention of motor function after the 3'-end extension of the pRNA, and the ease of RNA dimer, trimer, and hexamer manipulation with desired shape and size make this RNA-containing motor a promising tool for drug and gene delivery and for use in nanodevices.