Faculty Bibliography

Ribozymes are potential therapeutic agents which suppress specific genes in disease-affected cells. Ribozymes have high substrate cleavage efficiency, yet their medical application has been hindered by RNA degradation, aberrant cell trafficking, or misfolding when fused to a carrier. In this study, we constructed a chimeric ribozyme escorted by the motor pRNA of bacteriophage phi29 to achieve proper folding and enhanced stability. A pRNA molecule contains an interlocking loop domain and a 5'/3' helical domain, which fold independently of one another. When a ribozyme is connected to the helical domain, the chimeric pRNA/ribozyme reorganizes into a circularly permuted form, and the 5'/3' ends are relocated and buried in the original 71'/75' positions. Effective silencing of the anti-apoptotic gene survivin by an appropriately designed chimeric ribozyme, as demonstrated at mRNA and protein levels, led to programmed cell death in various human cancer cell lines, including breast, prostate, cervical, nasopharyngeal, and lung, without causing significant non-specific cytotoxicity. Through the interlocking interaction of right and left loops, monomer pRNA/ribozyme chimeras can be incorporated into multi-functional dimer, trimer and hexamer complexes for specific gene delivery. Using the phi29 motor pRNA as an escort may revive the ribozyme's strength in medical application.

By utilizing RNA nanotechnology, we engineered both therapeutic siRNA and a receptor-binding RNA aptamer into individual pRNAs of phi29's motor. The RNA building block harboring siRNA or other therapeutic molecules was fabricated subsequently into a trimer through the interaction of engineered right and left interlocking RNA loops. The incubation of the protein-free nanoscale particles containing the receptor-binding aptamer or other ligands resulted in the binding and co-entry of the trivalent therapeutic particles into cells, subsequently modulating the apoptosis of cancer cells and leukemia model lymphocytes in cell culture and animal trials. The use of such antigenicity-free 20-40 nm particles holds promise for the repeated long-term treatment of chronic diseases.

The application of small RNA in therapy has been hindered by the lack of an efficient and safe delivery system to target specific cells. Packaging RNA (pRNA), part of the DNA-packaging motor of bacteriophage phi29(Phi29), was manipulated by RNA nanotechnology to make chimeric RNAs that form dimers via interlocking right- and left-hand loops. Fusing pRNA with receptor-binding RNA aptamer, folate, small interfering RNA (siRNA), ribozyme, or another chemical group did not disturb dimer formation or interfere with the function of the inserted moieties. Incubation of cancer cells with the pRNA dimer, one subunit of which harbored the receptor-binding moiety and the other harboring the gene-silencing molecule, resulted in their binding and entry into the cells, and subsequent silencing of anti/proapoptotic genes. The chimeric pRNA complex was found to be processed into functional double-stranded siRNA by Dicer (RNA-specific endonuclease). Animal trials confirmed the suppression of tumorigenicity of cancer cells by ex vivo delivery. It has been reported [Shu, D., Moll, W.-D., Deng, Z., Mao, C., and Guo, P. (2004). Nano Lett. 4:1717-1724] that RNA can be used as a building block for bottom-up assembly in nanotechnology. The assembly of protein-free 25-nm RNA nanoparticles reported here will allow for repeated long-term administration and avoid the problems of short retention time of small molecules and the difficulties in the delivery of particles larger than 100 nm.

During assembly, bacterial virus phi29 utilizes a motor to insert genomic DNA into a preformed protein shell called the procapsid. The motor contains one twelve-subunit connector with a 3.6 nm central channel for DNA transportation, six viral-encoded RNA (packaging RNA or pRNA) and a protein, gp16, with unknown stoichiometry. Recent DNA-packaging models proposed that the 5-fold procapsid vertexes and 12-fold connector (or the hexameric pRNA ring) represented a symmetry mismatch enabling production of a force to drive a rotation motor to translocate and compress DNA. There was a discrepancy regarding the location of the foothold for the pRNA. One model [C. Chen and P. Guo (1997) J. Virol., 71, 3864-3871] suggested that the foothold for pRNA was the connector and that the pRNA-connector complex was part of the rotor. However, one other model suggested that the foothold for pRNA was the 5-fold vertex of the capsid protein and that pRNA was the stator. To elucidate the mechanism of phi29 DNA packaging, it is critical to confirm whether pRNA binds to the 5-fold vertex of the capsid protein or to the 12-fold symmetrical connector. Here, we used both purified connector and purified procapsid for binding studies with in vitro transcribed pRNA. Specific binding of pRNA to the connector in the procapsid was found by photoaffinity crosslinking. Removal of the N-terminal 14 amino acids of the gp10 protein by proteolytic cleavage resulted in undetectable binding of pRNA to either the connector or the procapsid, as investigated by agarose gel electrophoresis, SDS-PAGE, sucrose gradient sedimentation and N-terminal peptide sequencing. 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.

Bacterial virus phi29 is the most efficient in vitro DNA packaging system, with which up to 90% of the added DNA can be packaged into purified recombinant procapsid in vitro. The findings that phi29 virions can be assembled with the exclusive use of cloned gene products have bred a thought that phi29 has a potential to be a gene delivery vector since it is a nonpathogenic virus. gp12 of bacterial virus phi29 has been reported to be the anti-receptor that is responsible for binding the virus particle to the host cell. We cloned the gene coding gp12, overexpressed it in Escherichia coli, and purified the gene product to study the properties and functions of gp12 in virus assembly. According to SDS PloyAcrylamide Gel Electrophoresis (SDS-PAGE) analysis and N-terminal sequencing, recombinant gp12 isolated from E. coli had a molecular mass of 80 kDa, and 24 amino acids at N-terminal were cleaved after expression. The purified recombinant gp12 was incorporated into phi29 particles and converted the gp12-lacking assembly intermediates of phi29 into infectious virions in vitro. This purified protein gp12 was able to compete with infectious phi29 virions for binding to the host cell, thus inhibiting the infection by phi29. Scanning Transmission Electron Microscopy (STEM) analysis and sedimentation studies revealed that recombinant gp12 products were assembled into biologically active dimers. Analysis of the dose-response curve showed that 12 dimeric gp12 complexes were assembled onto viral particles and that each virion contained 24 copies of gp12 molecules. The results provide a basis for future research into bacteriophage-host interaction by modifying the anti-receptor protein. The ultimate goal is to re-target the bacteriophage to new host cells for the purpose of gene delivery.