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

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

Identification of copper death-associated molecular clusters and immunological profiles in rheumatoid arthritis

Objective: An analysis of the relationship between rheumatoid arthritis (RA) and copper death-related genes (CRG) was explored based on the GEO dataset. / Methods: Based on the differential gene expression profiles in the GSE93272 dataset, their relationship to CRG and immune signature were analysed. Using 232 RA samples, molecular clusters with CRG were delineated and analysed for expression and immune infiltration. Genes specific to the CRGcluster were identified by the WGCNA algorithm. Four machine learning models were then built and validated after selecting the optimal model to obtain the significant predicted genes, and validated by constructing RA rat models. / Results: The location of the 13 CRGs on the chromosome was determined and, except for GCSH. LIPT1, FDX1, DLD, DBT, LIAS and ATP7A were expressed at significantly higher levels in RA samples than in non-RA, and DLST was significantly lower. RA samples were significantly expressed in immune cells such as B cells memory and differentially expressed genes such as LIPT1 were also strongly associated with the presence of immune infiltration. Two copper death-related molecular clusters were identified in RA samples. A higher level of immune infiltration and expression of CRGcluster C2 was found in the RA population. There were 314 crossover genes between the 2 molecular clusters, which were further divided into two molecular clusters. A significant difference in immune infiltration and expression levels was found between the two. Based on the five genes obtained from the RF model (AUC = 0.843), the Nomogram model, calibration curve and DCA also demonstrated their accuracy in predicting RA subtypes. The expression levels of the five genes were significantly higher in RA samples than in non-RA, and the ROC curves demonstrated their better predictive effect. Identification of predictive genes by RA animal model experiments was also confirmed. / Conclusion: This study provides some insight into the correlation between rheumatoid arthritis and copper mortality, as well as a predictive model that is expected to support the development of targeted treatment options in the future

Specific delivery of therapeutic RNAs using bacteriophage phi29 pRNA as a vector

The clinical applications of small therapeutic RNAs including siRNA and ribozyme have been hindered by the lack of an efficient and safe delivery system that targets specific cells. In this study, packaging RNA (pRNA), a critical component of the DNA-packaging motor of bacteriophage phi29, was engineered by RNA nanotechnology to construct chimeric pRNA that contains gene silencing moiety (siRNA or ribozyme) or cell recognition moiety (folate or RNA aptamer). Chimeric pRNAs with inserted moieties retain the properties of forming dimers or trimers via interlocking interaction between the right- and left-hand loops. Incubation of cancer cells with a pRNA dimer, in which one subunit harbors folate as a cancer recognition moiety and the other contains siRNA against anti-apoptotic gene survivin, resulted in the binding and entry of this RNA dimer into the cancer cells and subsequent gene silencing. The chimeric pRNA/siRNA can be processed by Dicer and release double-stranded siRNA duplex to silence the target gene via the mechanism of RNA interference. The specific recognition of cancer cells and the anti-tumor activity of the pRNA dimer were further confirmed by ex vivo delivery in animal trials. In a trimeric pRNA complex, RNA aptamer against CD4 receptor mediated the binding and co-entry of the trivalent therapeutic particles into cells expressing CD4, and subsequently modulated the apoptosis of leukemia lymphocytes. The assembly of protein-free 25-nanometer RNA nanoparticles would 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 nanometer

Specific Delivery Of Therapeutic Rnas To Cancer Cells Via The Dimerization Mechanism Of Phi29 Motor Prna

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(φ29), 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. © Mary Ann Liebert, Inc

Controllable Self-Assembly Of Nanoparticles For Specific Delivery Of Multiple Therapeutic Molecules To Cancer Cells Using Rna Nanotechnology

By utilizing RNA nanotechnology, we engineered both therapeutic siRNA and a receptor-binding RNA aptamer into individual pRNAs of phi29\u27s 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. © 2005 American Chemical Society

Phi29 Prna Vector For Efficient Escort Of Hammerhead Ribozyme Targeting Survivin In Multiple Cancer Cells

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\u27s strength in medical application. ©2007 Landes Bioscience

A total of 5–20% sucrose gradient sedimentation of procapsid–pRNA complex treated before and after V8 treatment

<p><b>Copyright information:</b></p><p>Taken from "Binding of pRNA to the N-terminal 14 amino acids of connector protein of bacteriophage phi29"</p><p>Nucleic Acids Research 2005;33(8):2640-2649.</p><p>Published online 10 May 2005</p><p>PMCID:PMC1092275.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The closed rhombus is the procapsid–pRNA complex, while the open triangle is V8 pre-treated procapsid before adding pRNA (). The open square is V8-treated procapsid–pRNA complex ()

Specific binding of PpRNA I-i′ to connector demonstrated by UV crosslinking assay

<p><b>Copyright information:</b></p><p>Taken from "Binding of pRNA to the N-terminal 14 amino acids of connector protein of bacteriophage phi29"</p><p>Nucleic Acids Research 2005;33(8):2640-2649.</p><p>Published online 10 May 2005</p><p>PMCID:PMC1092275.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Lanes A–D, 10% SDS–PAGE autoradiographed pictures; lanes a–d, the same gel stained by Coomassie blue. Lane A, pRNA crosslinked to connector and then treated by RNase A; lane B, procapsid crosslinked to pRNA and then treated by RNase A; lane C, procapsid–pRNA complex without crosslinking, and then treated by RNase A; lane D, pRNA treated by RNase A. Lanes a, b, c and d correspond to A, B, C and D, respectively

A total of 10% SDS–PAGE to show the connector protein gp10 and procapsid treated before and after V8 cleavage

<p><b>Copyright information:</b></p><p>Taken from "Binding of pRNA to the N-terminal 14 amino acids of connector protein of bacteriophage phi29"</p><p>Nucleic Acids Research 2005;33(8):2640-2649.</p><p>Published online 10 May 2005</p><p>PMCID:PMC1092275.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Lane 1, procapsid alone; lane 2, procapsid cleaved by proteaseV8; lane 3, purified connector protein gp10 alone; lane 4, connector cleaved by V8; lane 5, connector–pRNA complex cleaved by V8. As noted in the text, in the procapsid, the C-terminus of gp10 is located at the wider end of the connector that is buried within the procapsid, while the N-terminus is located at the narrow end of the connector that is exposed to the solvent. Treatment of connector or procapsid with V8 resulted in different sizes of gp10, since V8 can cleave both the N- and C-terminus of gp10 of the free connector, but only the N-terminus of the gp10 that is buried within the procapsid. Gp8 is the capsid protein, while gp8.5 is the fiber protein of the procapsid

Engineering RNA for targeted siRNA delivery and medical application

RNA engineering for nanotechnology and medical applications is an exciting emerging research field. RNA has intrinsically defined features on the nanometer scale and is a particularly interesting candidate for such applications due to its amazing diversity, flexibility and versatility in structure and function. Specifically, the current use of siRNA to silence target genes involved in disease has generated much excitement in the scientific community. The intrinsic ability to sequence-specifically down-regulate gene expression in a temporally- and spatially-controlled fashion has led to heightened interest and rapid development of siRNA-based therapeutics. Though methods for gene silencing with high efficacy and specificity have been achieved in vitro, the effective delivery of nucleic acids to specific cells in vivo has been a hurdle for RNA therapeutics. This review covers different RNA-based approaches for diagnosis, prevention and treatment of human disease, with a focus on the latest developments of nonviral carriers of siRNA for delivery in vivo. The applications and challenges of siRNA therapy, as well as potential solutions to these problems, the approaches for using phi29 pRNA-based vectors as polyvalent vehicles for specific delivery of siRNA, ribozymes, drugs or other therapeutic agents to specific cells for therapy will also be addressed