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A controllable, 30-nm imitating DNA-packaging motor was constructed. The motor is driven by six synthetic ATP-binding pRNAs arranged as a hexamer, similar to the driving of a bolt with a hex nut. Conformational change and sequential action of the RNA, with its five-fold (viral capsid)/six-fold (pRNA hexamer) mismatch, could ensure continuous motion of the motor with ATP as energy.

In the presence of ATP and magnesium, a 5 μm 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 per ml of infectious virus.

The 3D structures of pRNA monomer, dimer and hexamer have been probed by photo affinity crosslinking, chemical modification interference, cryo-atomic force microscopy, and computer modeling¹. pRNA’s size and shape can be manipulated at will to form stable dimers and trimers². The motor can be turned off and turned on again³. The formation of ordered structural arrays of the motor complex and its components, retention of motor function after the 3’-end extension of the pRNA, and the ease of RNA manipulation make this RNA-containing motor a promising tool for use in nanodevices.

Related papers from this lab:
1. S. Hoeprich and P. Guo J Biol.Chem., 277:20794 (2002).
2. D. Shu, L. Huang and P. Guo. J. Nanoscience & Nanotechnology 3:1 (2003).
3. D. Shu and P. Guo. J.Biol.Chem., 278:7119 (2003).
4. C. Chen and P. Guo. J.Virol., 71:3864 (1997).

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Biological macromolecules including protein and DNA have been reported as powerful nanostructuring tools and nanodevices. However, RNA has remained poorly explored by nanoscientists. The attractive features of RNA for designing bionanostructures include its versatile structure, the relative ease with which it can be produced and manipulated, and its programmable self-assembly properties. pRNA with its unusual structural and functional features is an ideal starting material for RNA nanotechnology. By making complementary mutations in four nucleotides from pRNA’s left and right loops, we engineered pRNA molecules that could form stable dimers and trimers in a protein-free environment with very high efficiency. Since the pairing of the ends of pRNA does not hinder the assembly of hexamers, arrays, and other structures, modifications including extensions, deletions and circular permutations of this domain can give rise to defined self-assembled nanostructures of various sizes and shapes. These building blocks were capable of self-assembling into rods, triangles or three dimensional arrays with micrometer size dimensions. Arrays were found to be highly stable in a variety of environmental conditions. Our results demonstrate that RNA has the potential to serve as versatile building block in nanobiotechnology.

Related papers from this lab:
1. Shu D, Moll D, Deng Z, Mao C, and Guo P. 2004. Bottom-up assembly of RNA arrays and superstructures as potential parts in nanotechnology. Nano Letters 4: 1717-1724.
2. Shu D, Huang LP, Hoeprich S, & Guo P (2003): J. Nanosci. Nanotechnol. 3: 295-302.

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Both siRNA and RNA enzymes known as ribozymes show significant potential to be used for the treatment and/or prevention of diseases in plants, humans and animals. Although it has long been established that hammerhead ribozymes could cleave specific RNA in the test tube, efficiency has traditionally been dramatically reduced due to their misfolding and degradation by exonucleases. The phi29 vector effectively transports and protects the hammerhead ribozyme, and the assembled complex has already been proven successful in targeting the Hepatitis B virus (see figure).

Related papers from this lab:
1. Guo, S., Shu, D., Simon, M., Guo, P., 2003. Gene cloning, purification and stoichiometry quantification of phi29 anti-receptor gp12 with potential use as special ligand for gene delivery . Gene 315 (2003) 145-152.
2. Hoeprich, S., Zhou, Q. G. S., Shu, D., Qi, G., Wang, Y., Guo, P., 2003. Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus. Gene Therapy 10(15), 1258-1267.

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The treatment of cancers has long been hindered by the lack of an efficient polyvalent targeting and delivery system that can simultaneously allow for: 1. Targeting of specific receptors on the surface of human cancer cells, 2. Safe cellular entry, and 3.) Delivery of the necessary variety of therapeutic molecules to cancer cells. The goal of this project is to further refine an in vivo gene delivery system for cancer therapy based on the in vitro assembly system of bacteriophage phi29. Advances in packaging and vector assembly has been accomplished in the following areas of interest: 1. A highly efficient in vitro viral packaging system for the encapsidation of specific and non-specific DNA has been developed, 2.) Dual plasmid vectors with the expression of reporter genes in both prokaryotic and eukaryotic cells have been constructed, 3. A new strategy for complete intracellular immunization to viral infection has been developed, and 4. Successful early testing of delivery particles carrying specific ligands to recognize receptors on the surface of cells from cancerous cell lines suggests that specific targeting in a clinical setting will be possible in the future.

Related papers from this lab:
1. Guo, S., Shu, D., Simon, M., Guo, P., 2003. Gene cloning, purification and stoichiometry quantification of phi29 anti-receptor gp12 with potential use as special ligand for gene delivery . Gene 315 (2003) 145-152.

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The capsid of dsDNA bacterial viruses contains a six-fold symmetrical DNA-translocating machine or connector embedded in a protein shell with five-fold rotational symmetry. Our laboratory’s model suggests that the relative motion of the two symmetrical, mismatched rings provides the driving force for DNA translocation. Through a mechanism analogous to the six cylinders of a car’s engine, sequential action would turn the motor. The finding that six copies of pRNA were bound to the connector and worked sequentially provides important support to this model. To make two rings rotate relatively, at least one additional component is needed to provide a propelling force. We found that the packaging of phi29 DNA requires ATP as an driving energy source and phi29 pRNA binds ATP¹. RNA is required to fuel ATPase activity during DNA packaging. This is the first recorded instance of the interaction of RNA with ATP. We have also found that RNA is part of the ATPase. Alternative conformation changes of pRNA would provide the physical force for DNA translocation. This project’s purpose is to study how ATP can cause conformational change in pRNA.

Related papers from this lab:
1. D. Shu and P. Guo. J.Biol.Chem., 278:7119 (2003).