Bacteriophage Phi Research Articles

Dynamics of the Translocation Step Measured in Individual DNA Polymerase Complexes

Complexes formed between the bacteriophage phi29 DNA polymerase (DNAP) and DNA fluctuate between the pre-translocation and post-translocation states on the millisecond time scale. These fluctuations can be directly observed with single-nucleotide precision in real-time ionic current traces when individual complexes are captured atop the α-hemolysin nanopore in an applied electric field. We recently quantified the equilibrium across the translocation step as a function of applied force (voltage), active-site proximal DNA sequences, and the binding of complementary dNTP. To gain insight into the mechanism of this step in the DNAP catalytic cycle, in this study, we have examined the stochastic dynamics of the translocation step. The survival probability of complexes in each of the two states decayed at a single exponential rate, indicating that the observed fluctuations are between two discrete states. We used a robust mathematical formulation based on the autocorrelation function to extract the forward and reverse rates of the transitions between the pre-translocation state and the post-translocation state from ionic current traces of captured phi29 DNAP-DNA binary complexes. We evaluated each transition rate as a function of applied voltage to examine the energy landscape of the phi29 DNAP translocation step. The analysis reveals that active-site proximal DNA sequences influence the depth of the pre-translocation and post-translocation state energy wells and affect the location of the transition state along the direction of the translocation.

Fluorogenic RNA Nanoparticles for Monitoring RNA Folding and Degradation in Real Time in Living Cells

Due to the discovery of more and more roles of cellular noncoding RNAs, the approaches for introducing RNAs including small interfering RNA (siRNA), micro RNA (miRNA), ribozyme, and riboswitch into cells for regulating cell life cycle and for the treatment of diseases have become routine practice. The understanding of RNA folding, degradation, and intracellular half-life after entering the cell is an intriguing question in biology and pharmacology. Currently, methods to detect RNA folding, degradation, and half-life in real time within the cell is extremely challenging. The common assay method to measure RNA half-life and degradation in vivo is the use of radioactive markers or fluorescence RNA labeling. The challenge is, after RNA becomes degraded or misfolded, the isotope or the fluorescence is still present in the cell, thus the signals are not a true indication of the presence of the RNA in the cell. The alternate method commonly used to measure RNA life is to isolate RNA from cells and distinguish between intact and degraded RNA by gel, chromatography, or capillary electrophoresis. However, when a cell is breaking down, ribonucleases (RNases) will be released from cell compartments, and degradation of small RNA in cell lysates occurs immediately after cell lysis. Here we report a method to monitor RNA degradation in real time in living cells using fluorogenic RNA in combination with RNA nanotechnology (Guo, 2010; Guo et al., 2012). The RNA aptamer that binds malachite green (MG), the ribozyme that cleaves the hepatitis virus genome, and a siRNA for firefly luciferase were all fused to the bacteriophage phi29 packaging RNA (pRNA) 3-way junction (3WJ) motif to generate RNA nanoparticles. The MG aptamer, the hepatitis B virus ribozyme, and the luciferase siRNA all retained their function independently after fusion into the nanoparticles. When the RNA nanoparticle is degraded, denatured, or misfolded, the fluorescence disappears. MG, which is not fluorescent by itself, is capable of binding to its aptamer and emitting fluorescent light only if the RNA remains folded in the correct conformation. Therefore, the MG aptamer fluorescence (in the presence of MG dye) can be used as a measure of the degradation and folding of RNA nanoparticles, the siRNA, the aptamer, and the ribozyme in the cell in real time using epifluorescence microscopy and fluorescence spectroscopy without lysing the cells. We show that the half-life (t½) of the electroporated MG aptamer containing RNA nanoparticle was 4.3 hours after electroporation into cells.

Physical Virology: Direct Measurement of Phage phi29 Stiffness Provides Evidence of Internal Pressure (Small 15/2012)

A 3D AFM topography rendition of a phage phi29 (red) adsorbed on mica (blue) is shown by P. J. de Pablo and coworkers, where part of the viral DNA (yellow) has been ejected through the tail. The phi29 bacteriophage translocates part of its DNA into the host by releasing the elastic energy arising from the internal pressure created during the DNA-packing process. Pushing with the AFM tip on the phage enables the direct measurement of the stiffness to estimate its internal pressure, just as a tyre may be pressed with fingers.

Construction of Semisynthetic DNA–Protein Conjugates with Phi X174 Gene-A* Protein

DNA-protein conjugates have frequently been used as versatile molecular tools for a variety of applications in biotechnology to harness synergistic effects of DNA and protein functions. With applications for DNA-protein conjugates growing, easy-to-use and economical methods for the synthesis of DNA-protein conjugates are required. In this study, we developed a method for site-specific labeling of single-stranded DNA (ssDNA) to a recombinant protein of interest (POI) through the Gene-A* protein (Gene-A*) from bacteriophage phi X174, without any chemical modifications of ssDNA. Gene-A* protein is an enzyme that site-selectively cleaves an oligodeoxyribonucleotide (ODN) containing a Gene-A* recognition sequence, at which point a tyrosine residue of Gene-A* is bonded to the 5'-phosphoryl group of the cleavage site via a stable phosphotyrosine linkage. Here, we constructed three kinds of recombinant proteins fused to Gene-A*: N-terminally Gene-A*-fused enhanced green fluorescent protein (EGFP), C-terminally Gene-A*-fused EGFP, and N-terminally Gene-A*-fused firefly luciferase (FLuc). The reaction yields of DNA-protein conjugation catalyzed by the Gene-A* moiety reached 80-90% in the three proteins, and kinetic study revealed that the reaction achieved a steady state after 10 min. Moreover, dot blot analyses were performed to evaluate the hybridization and aptamer-forming ability of ssDNA conjugated to the Gene-A* moiety of a recombinant Gene-A*-FLuc protein. This study demonstrated that a strategy using recombinant proteins fused to Gene-A* could offer a versatile, rapid, easy-to-use, and economical platform for producing DNA-protein conjugates.

Direct Measurement of Phage phi29 Stiffness Provides Evidence of Internal Pressure

Using AFM nanoindentation experiments, DNA-full phi29 phage capsids are shown to be stiffer than when empty. The presence of counterions softens full viruses in a reversible manner, indicating that pressure originates from the confined DNA. A finite element analysis of the experiments provides an estimate of the pressure of ∼40 atm inside the capsid, which is similar to theoretical predictions.

Real-Time Sensing and Discrimination of Single Chemicals Using the Channel of Phi29 DNA Packaging Nanomotor

A highly sensitive and reliable method to sense and identify a single chemical at extremely low concentrations and high contamination is important for environmental surveillance, homeland security, athlete drug monitoring, toxin/drug screening, and earlier disease diagnosis. This article reports a method for precise detection of single chemicals. The hub of the bacteriophage phi29 DNA packaging motor is a connector consisting of 12 protein subunits encircled into a 3.6 nm channel as a path for dsDNA to enter during packaging and to exit during infection. The connector has previously been inserted into a lipid bilayer to serve as a membrane-embedded channel. Herein we report the modification of the phi29 channel to develop a class of sensors to detect single chemicals. The lysine-234 of each protein subunit was mutated to cysteine, generating 12-SH ring lining the channel wall. Chemicals passing through this robust channel and interactions with the SH group generated extremely reliable, precise, and sensitive current signatures as revealed by single channel conductance assays. Ethane (57 Da), thymine (167 Da), and benzene (105 Da) with reactive thioester moieties were clearly discriminated upon interaction with the available set of cysteine residues. The covalent attachment of each analyte induced discrete stepwise blockage in current signature with a corresponding decrease in conductance due to the physical blocking of the channel. Transient binding of the chemicals also produced characteristic fingerprints that were deduced from the unique blockage amplitude and pattern of the signals. This study shows that the phi29 connector can be used to sense chemicals with reactive thioesters or maleimide using single channel conduction assays based on their distinct fingerprints. The results demonstrated that this channel system could be further developed into very sensitive sensing devices.

Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling

The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (H(T) and H(L)) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which H(T) and H(L) stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.

Single Molecule Studies of the Effect of Spermidine on DNA Mechanics and Viral DNA Packaging

Polyamine ions such as spermidine 3+ , along with monovalent and divalent salt ions, screen the negatively charged backbone of dsDNA and thereby facilitate processes in which DNA is confined in small spaces, such as viral DNA packaging. We use optical tweezers to directly manipulate single DNA molecules and have made preliminary measurements of the effect of spermidine on DNA elasticity, condensation, and viral packaging. We determine the concentration of spermidine 3+ at which dsDNA condenses in the presence of Mg 2+ and Na + and report a monotonic increase in stretch modulus and decrease in persistence length at incremental spermidine concentrations up to the concentration at which dsDNA condenses. We also discuss the effect of spermidine on DNA packaging in bacteriophage phi29.

Single Molecule Studies of Bacteriophage phi29 DNA Terminal Protein Interactions and their Role in DNA Packaging

The assembly of a functional DNA-packaging complex is an essential process in the lifecycle of the tailed double-stranded DNA bacteriophage, phi29; however a detailed understanding of the assembly pathway is still missing. Phi29 and its relatives, as well as some Adenoviruses, possess linear double stranded DNA genomes with proteins covalently linked to the 5’ terminal ends of each strand. In addition to the terminal protein's (TP) main role in protein-primed DNA replication, the phi29 TP, gp-3, appears to play an essential role in the formation of the mature DNA packaging substrate. Evidence suggests gp3 mediates the formation of DNA loops which are subsequently bound by the motor protein, gp16, and supercoiled to form the mature packaging substrate. We have developed a single molecule optical tweezers assay that allows us to measure gp3-DNA and gp3-gp3 interactions and probe their role in the initiation of motor-driven viral DNA packaging. Here we present the direct observation of strong sequence-specific gp3-phi29DNA interactions as well as evidence for preferential binding of left-end gp3 to a region near the left terminal end of the genome, with the later suggesting a mechanism by which the virus may preferentially package the left end of its genome in vivo. We will also discuss efforts to determine whether gp16 in isolation, apart from the viral capsid portal complex, can act as a DNA translocase.

Complete Characterization of the Mechanochemical Cycle of a Homomeric Ring ATPase

Understanding how multimeric ring ATPases couple chemical transitions to mechanical motion and coordinate their subunits is essential for elucidating the functions of these important molecular machines. Here we used high-resolution optical tweezers to study one such ATPase - the bacteriophage phi29 DNA packaging motor. This pentameric motor is known to translocate DNA by cycling through a dwell phase, in which subunits load ATPs, and a burst phase, in which 10 base pairs of DNA are packaged in four 2.5-base-pair steps. By monitoring packaging in buffers containing various nucleotides and nucleotide analogs, we determined the exact timing of chemical transitions (nucleotide hydrolysis and product release) with respect to the mechanical phases of the motor's cycle. Our results reveal an intricate coordination mechanism with multiple levels of communication between neighboring subunits. ADP release and ATP binding occur in a concerted alternating fashion, with ATP binding to one subunit facilitating the release of ADP in the following subunit. ATP hydrolysis also appears to occur sequentially around the ring through an intersubunit allosteric effect. Moreover, our data suggest that all five subunits of the ring bind and hydrolyze ATP. Only four out of the five hydrolysis events are coupled to DNA translocation, while the fifth subunit consumes ATP to fulfill a regulatory role, resetting the motor for the next mechanochemical cycle. The phi29 motor is able to tolerate a single inactive subunit and still function, albeit at a much slower pace. These new findings allow us to present the most complete mechanochemical model of a homomeric ring ATPase to date, which should provide insight into the operating principles of other ring-shaped ATPases.

Global Structure of a Three-Way Junction in a phi29 Packaging RNA Dimer determined using Site-Directed Spin Labeling

The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component, called the packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA 3-way junction, which bridges binding sites for the motor ATPase and the procapsid. The studies were carried out in a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals (R5) were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 inter-R5 distances spanning the 3-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select approximately 3,600 viable 3-way junction models from a pool of 65 billion. The results reveal a similar conformation among all the viable models, with two of the helices (HT and HL) adopting an acute bend. This contrasts to a recently reported pRNA tetramer crystal structure, in which HT and HL stack onto each other linearly. The studies reveal versatility in pRNA conformations that may be beneficial to packaging motor function, and establish a new venue for mapping global structures of complex RNA molecules.

Sequential action of ATPase, ATP, ADP, Pi and dsDNA in procapsid-free system to enlighten mechanism in viral dsDNA packaging

Many cells and double-stranded DNA (dsDNA) viruses contain an AAA+ ATPase that assembles into oligomers, often hexamers, with a central channel. The dsDNA packaging motor of bacteriophage phi29 also contains an ATPase to translocate dsDNA through a dodecameric channel. The motor ATPase has been investigated substantially in the context of the entire procapsid. Here, we report the sequential action between the ATPase and additional motor components. It is suggested that the contact of ATPase to ATP resulted in its conformational change to a higher binding affinity toward dsDNA. It was found that ATP hydrolysis led to the departure of dsDNA from the ATPase/dsDNA complex, an action that is speculated to push dsDNA to pass the connector channel. Our results suggest that dsDNA packaging goes through a combined effort of both the gp16 ATPase for pushing and the channel as a one-way valve to control the dsDNA translocation direction. Many packaging models have previously been proposed, and the packaging mechanism has been contingent upon the number of nucleotides packaged per ATP relative to the 10.5 bp per helical turn for B-type dsDNA. Both 2 and 2.5 bp per ATP have been used to argue for four, five or six discrete steps of dsDNA translocation. Combination of the two distinct roles of gp16 and connector renews the perception of previous dsDNA packaging energy calculations and provides insight into the discrepancy between 2 and 2.5 bp per ATP.

High-throughput analysis of growth differences among phage strains

Although methods such as spectrophotometry are useful for identifying growth differences among bacterial strains, it is currently difficult to similarly determine whether bacteriophage strains differ in growth using high throughput methods. Here we use automated spectrophotometry to develop an in vitro method for indirectly distinguishing fitness (growth) differences among virus strains, based on direct measures of their infected bacterial hosts. We used computer simulations of a mathematical model for phage growth to predict which features of bacterial growth curves were best associated with differences in growth among phage strains. We then tested these predictions using the in vitro method to confirm which of the inferred viral growth traits best reflected known fitness differences among genotypes of the RNA phage phi-6, when infecting a Pseudomonas syringae host. Results showed that the inferred phage trait of time-to-extinction (time required to drive bacterial density below detectable optical density) reliably correlated with genotype rankings based on absolute fitness (phage titer per ml). These data suggested that the high-throughput analysis was valuable for identifying growth differences among virus strains, and that the method may be especially useful for high throughput analyses of fitness differences among phage strains cultured and/or evolved in liquid (unstructured) environments.

Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics

RNA nanoparticles can potentially be used to treat cancers and viral infection but their instability has hindered their therapeutic applications. The lack of covalent linkage or cross-linking in the nanoparticles causes dissociation in vivo. Here we show the assembly of thermodynamically stable three-way junction (3WJ) of the motor pRNA of bacteriophage phi29 from 3–6 pieces of RNA oligomers without the use of metal salts to form stable multifunctional nanoparticles. Each RNA oligomer contains either a receptor-binding ligand, aptamer, siRNA or ribozyme functional module. When mixed together, they self-assemble into tri-stars nanoparticles with a 3WJ core. The nanoparticles are resistant to 8M urea denaturation, stable in serum and remain intact at extremely low concentrations. The modules remain functional in vitro and in vivo, suggesting the 3WJ core can be used as a platform for building a variety of multifunctional nanoparticles. Of the 25 different 3WJ motifs evaluated, only one other motif in biological RNA shares similar characteristics as the phi29 3WJ.

Three reversible and controllable discrete steps of channel gating of a viral DNA packaging motor

The channel of the viral DNA packaging motor allows dsDNA to enter the protein procapsid shell during maturation and to exit during infection. We recently showed that the bacteriophage phi29 DNA packaging motor exercises a one-way traffic property using a channel as a valve for dsDNA translocation. This raises a question of how dsDNA is ejected during infection if the channel only allows the dsDNA to travel inward. We proposed that DNA forward or reverse travel is controlled by conformational changes of the channel. Here we reported our direct observation that the channel indeed exercises conformational changes by single channel recording at a single-molecule level. The changes were induced by high electrical voltage, or by affinity binding to the C-terminal wider end located within the capsid. Novel enough, the conformational change of the purified connector channel exhibited three discrete gating steps, with a size reduction of 32% for each step. We investigated the role of the terminal and internal loop of the channel in gating by different mutants. The step-wise conformational change of the channel was also reversible and controllable, making it an ideal nano-valve for constructing a nanomachine with potential applications in nanobiotechnology and nanomedicine.

Assembly of Therapeutic pRNA-siRNA Nanoparticles Using Bipartite Approach

The 117-nucleotide (nt) RNA, called the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor, has been shown to be an efficient vector for the construction of RNA nanoparticles for the delivery of small interfering RNA (siRNA) into specific cancer or viral-infected cells. Currently, chemical synthesis of 117-nt RNA is not feasible commercially. In addition, labeling at specific locations on pRNA requires the understanding of its modular organization. Here, we report multiple approaches for the construction of a functional 117-base pRNA using two synthetic RNA fragments with variable modifications. The resulting bipartite pRNA was fully competent in associating with other interacting pRNAs to form dimers, as demonstrated by the packaging of DNA via the nanomotor and the assembly of phi29 viruses in vitro. The pRNA subunit assembled from bipartite fragments harboring siRNA or receptor-binding ligands were equally competent in assembling into dimers. The subunits carrying different functionalities were able to bind cancer cells specifically, enter the cell, and silence specific genes of interest. The pRNA nanoparticles were subsequently processed by Dicer to release the siRNA embedded within the nanoparticles. The results will pave the way toward the treatment of diseases using synthetic pRNA/siRNA chimeric nanoparticles.

Pharmacological Characterization of Chemically Synthesized Monomeric phi29 pRNA Nanoparticles for Systemic Delivery

Previous studies have shown that the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor folds into a compact structure, constituting a RNA nanoparticle that can be modularized with functional groups as a nanodelivery system. pRNA nanoparticles can also be self-assembled by the bipartite approach without altering folding property. The present study demonstrated that 2'-F-modified pRNA nanoparticles were readily manufactured through this scalable bipartite strategy, featuring total chemical synthesis and permitting diverse functional modularizations. The RNA nanoparticles were chemically and metabolically stable and demonstrated a favorable pharmacokinetic (PK) profile in mice (half-life (T(1/2)): 5-10 hours, clearance (Cl): <0.13 l/kg/hour, volume of distribution (V(d)): 1.2 l/kg). It did not induce an interferon (IFN) response nor did it induce cytokine production in mice. Repeat intravenous administrations in mice up to 30 mg/kg did not result in any toxicity. Fluorescent folate-pRNA nanoparticles efficiently and specifically bound and internalized to folate receptor (FR)-bearing cancer cells in vitro. It also specifically and dose-dependently targeted to FR(+) xenograft tumor in mice with minimal accumulation in normal tissues. This first comprehensive pharmacological study suggests that the pRNA nanoparticle had all the preferred pharmacological features to serve as an efficient nanodelivery platform for broad medical applications.

Targeted Delivery of Mutant Tolerant Anti-Coxsackievirus Artificial MicroRNAs Using Folate Conjugated Bacteriophage Phi29 pRNA

BackgroundMyocarditis is the major heart disease in infants and young adults. It is very commonly caused by coxsackievirus B3 (CVB3) infection; however, no specific treatment or vaccine is available at present. RNA interference (RNAi)-based anti-viral therapy has shown potential to inhibit viral replication, but this strategy faces two major challenges; viral mutational escape from drug suppression and targeted delivery of the reagents to specific cell populations.Methodology/Principal FindingsIn this study, we designed artificial microRNAs (AmiRs) targeting the 3′untranslated region (3′UTR) of CVB3 genome with mismatches to the central region of their targeting sites. Antiviral evaluation showed that AmiR-1 and AmiR-2 reduced CVB3 (Kandolf and CG strains) replication approximately 100-fold in both HeLa cells and HL-1 cardiomyoctes. To achieve specific delivery, we linked AmiRs to the folate-conjugated bacterial phage packaging RNA (pRNA) and delivered the complexes into HeLa cells, a folate receptor positive cancer cells widely used as an in vitro model for CVB3 infection, via folate-mediated specific internalization. We found that our designed pRNA-AmiRs conjugates were tolerable to target mutations and have great potential to suppress viral mutational escape with little effect on triggering interferon induction.Conclusion/SignificanceThis study provides important clues for designing AmiRs targeting the 3′UTR of viral genome. It also proves the feasibility of specific deliver of AmiRs using conjugated pRNA vehicles. These small AmiRs combined with pRNA-folate conjugates could form a promising system for antiviral drug development.

INTERACTION OF VIRAL AND BACTERIAL LYSOZYMES WITH ERWINIA AMYLOVORA AND THEIR INHIBITION BY A BACTERIAL PROTEIN

Viral lysozymes can interact with Erwinia amylovora, and their genes may control fire blight when expressed in the pathogen. We have investigated lysozymes from E. amylovora phages and from genomes of several Erwinia species. With chloroform-treated cells as substrate, the highest lytic activity was found for viral lysozymes. Fusion proteins with the (phi)Ea1h lysozyme, such as addition of a His-tag reduced lysis efficiency significantly. For large fusions at the N- or C-terminus lytic activity was completely lost. When a protein fused to glutathione S-transferase was cleaved off with a specific protease, the activity of the viral lysozyme was restored. Intact lysozyme from E. amylovora phage ΦEa1h had a similar lytic activity as E. coli phage T4 lysozyme. Viable cells of E. amylovora were not inhibited by external addition of lysozyme from sonicated cell extracts in assays with (phi)Ea1h lysozyme and T4 lysozyme. The cellular expression of a viral lysozyme gene can be toxic for a bacterial host. The gene was therefore expressed in yeast as safe environment for its secretion. Lysozyme-specific antibodies detected the protein in culture supernatants of transgenic yeast. When E. amylovora was transformed with a viral lysozyme gene, the cells were killed after induction by IPTG. Transfer of a lyz-gene into E. amylovora can thus strongly reduce the viability of the pathogen. The (phi)Ea1h lysozyme was inhibited by a cellular protein from E. amylovora related to an inhibitor for vertebrate lysozymes of E. coli. The lytic activity of lysozyme from phage (phi)Ea104 and (phi)Ea116 was not affected by the enzyme inhibitor.

RNA nanoparticles come of age

RNA nanoparticles come of age