Bacteriophage Phi29 DNA Packaging Motor Research Articles

Utilizing RNA nanotechnology to construct negatively charged and ultrasound-responsive nanodroplets for targeted delivery of siRNA

Ultrasound nanodroplets (NDs) have been reported as a promising nanocarrier for siRNA delivery depending on its unique strengths of sonoporation. Presently, common means for NDs-mediated siRNA delivery is through electrostatic interaction, but challenges like cationic toxicity still exist. In this study, we demonstrated a novel strategy to construct negatively charged and ultrasound (US)-responsive O-carboxymethyl chitosan (O-CMS) NDs as a siRNA targeted delivery system through three-way junction of bacteriophage phi29 DNA packaging motor (3WJ-pRNA) nanotechnology. 39nt A10-3.2 aptamer targeting prostate specific membrane antigen (PSMA) and 21nt siRNA against cationic amino acid transporter 1 (siCAT-1) were annealed to 3WJ-pRNA scaffold via complementation with an extended sequence. The cholesterol molecule attached to one branch facilitates the 3WJ-pRNA nanoparticles anchoring onto NDs. The desired O-CMS NDs with siRNA-loading and RNA-aptamer modification (A10-3.2/siCAT-1/3WJ-NDs) were successfully prepared, which were with spherical shapes, core–shell structures and uniform in sizes (198 nm with PDI 0.3). As a main proportion of shell, O-CMC showed a certain anti-tumor effects. In vitro studies demonstrated that A10-3.2/siCAT-1/3WJ-NDs exhibited good contrast-enhanced US imaging, buffering capacity and high bio-safety, were able to deliver siCAT-1 to PSMA-overexpressed prostate cancer cells under US irradiation, thus silence the CAT-1 expression, and consequently suppressing 22RV1 cell proliferation and migration. Taken overall, our findings provide a promising strategy to develop negatively charged and US-responsive NDs for tumor-targeted siRNA delivery.

Insertion of channel of phi29 DNA packaging motor into polymer membrane for high-throughput sensing

The connector channel of bacteriophage phi29 DNA packaging motor has been inserted into the lipid bilayer membrane and has shown potential for the sensing of DNA, RNA, chemicals, peptides, and antibodies. Properties such as high solubility and large channel size have made phi29 channel an advantageous system for those applications; however, previously studied lipid membranes have short lifetimes, and they are frangible and unstable under voltages higher than 200 mV. Thus, the application of this lipid membrane platform for clinical applications is challenging. Here we report the insertion of the connector into the stable polymer membrane in MinION flow cell that contains 2048 wells for high-throughput sensing by the liposome-polymer fusion process. The successful insertion of phi29 connector was confirmed by a unique gating phenomenon. Peptide translocation through the inserted phi29 connector was also observed, revealing the potential of applying phi29 connector for high-throughput peptide sensing.

RNA nanoparticle distribution and clearance in the eye after subconjunctival injection with and without thermosensitive hydrogels

Thermodynamically and chemically stable RNA nanoparticles derived from the three-way junction (3WJ) of the pRNA from bacteriophage phi29 DNA packaging motor were examined previously for ocular delivery. It was reported that, after subconjunctival injection, RNA nanoparticles with tri-way shape entered the corneal cells but not the retinal cells, whereas particle with four-way shape entered both corneal and retinal cells. The present study evaluated ocular delivery of RNA nanoparticles with various shapes and sizes, and assessed the effect of thermosensitive hydrogels (poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid); PLGA-PEG-PLGA) for increasing the retention of RNA nanoparticles in the eye. Fluorescence imaging of mouse eyes and fluorescence microscopy of dissected eye tissues from the conjunctiva, cornea, retina, and sclera were performed to determine the distribution and clearance of the nanoparticles in the eyes after subconjunctival injection in vivo. RNA nanoparticles entered the cells of the conjunctiva, cornea, retina, and sclera after subconjunctival delivery. The clearance of RNA pentagon was slower than both RNA square and triangle of the same designed edge length (10nm) in the eye, and the clearance of RNA squares of the longer edge lengths (10 and 20nm) was slower than RNA square of the shorter edge length (5nm), thus indicating that the size could affect ocular pharmacokinetics of the nanoparticles. At 24h after the injection, approximately 6–10% of the fluorescence signal from the larger nanoparticles in the study (RNA square of 20nm edge length and RNA pentagon of 10nm edge length) remained in the eye, and up to 70% of the retinal cells contained the nanoparticles. The results suggest that the larger nanoparticles were “gulped” in conjunctival, corneal, retinal, and scleral cells, similar to the behavior observed in macrophages. Additionally, the combination of RNA nanoparticles with the thermosensitive polymers increased the retention of the nanoparticles in the eye.

Dual Tumor-Targeting Nanocarrier System for siRNA Delivery Based on pRNA and Modified Chitosan.

Highly specific and efficient delivery of siRNA is still unsatisfactory. Herein, a dual tumor-targeting siRNA delivery system combining pRNA dimers with chitosan nanoparticles (CNPPs) was designed to improve the specificity and efficiency of siRNA delivery. In this dual delivery system, folate-conjugated and PEGylated chitosan nanoparticles encapsulating pRNA dimers were used as the first class of delivery system and would selectively deliver intact pRNA dimers near or into target cells. pRNA dimers simultaneously carrying siRNA and targeting aptamer, the second class of delivery system, would specifically deliver siRNA into the target cells via aptamer-mediated endocytosis or proper particle size. To certify the delivering efficiency of this dual system, CNPPs, pRNA dimers alone, chitosan nanoparticles containing siRNA with folate conjugation and PEGylation (CNPS), and chitosan nanoparticles containing pRNA dimers alone (CN) were first prepared. Then, we observed that treatment with CNPPs resulted in increased cellular uptake, higher cell apoptosis, stronger cell cytotoxicity, and more efficacious gene silencing compared to the other three formulations. Higher accumulation of siRNA in the tumor site, stronger tumor inhibition, and longer circulating time were also observed with CNPPs compared to other formulations. In conclusion, this dual nanocarrier system showed high targeting and favorable therapeutic efficacy both in vitro and in vivo. Thereby, a new approach is provided in this study for specific and efficient delivery of siRNA, which lays a foundation for the development of pRNA hexamers, which can simultaneously carry six different substances.

Fingerprinting and Differentiation of Small Proteins with a Large Channel of Bacteriophage PHI29 DNA Packaging Motor

Fingerprinting and Differentiation of Small Proteins with a Large Channel of Bacteriophage PHI29 DNA Packaging Motor

RNA Nanoparticle-Based Targeted Therapy for Glioblastoma through Inhibition of Oncogenic miR-21

Targeted inhibition of oncogenic miRNA-21 has been proposed to treat glioblastoma by rescuing tumor suppressors, PTEN and PDCD4. However, systemic delivery of anti-miR-21 sequences requires a robust and efficient delivery platform to successfully inhibit this druggable target. Three-way-junction (3WJ)-based RNA nanoparticles (RNP), artificially derived from pRNA of bacteriophage phi29 DNA packaging motor, was recently shown to target glioblastoma. Here, we report that multi-valent folate (FA)-conjugated 3WJ RNP constructed to harbor anti-miR-21 LNA sequences (FA-3WJ-LNA-miR21) specifically targeted and delivered anti-miR-21 LNA and knocked down miR-21 expression in glioblastoma cells invitro and invivo with favorable biodistribution. Systemically injected FA-3WJ-LNA-miR21 RNP efficiently rescued PTEN and PDCD4, resulting in glioblastoma cell apoptosis and tumor growth regression. Overall survival rate was also significantly improved by FA-3WJ-LNA-miR21 RNP. These results are indicative of the clinical benefit of FA-3WJ RNP-based gene therapy for the successful targeted therapy of developing and even recurring glioblastoma.

Using Planar Phi29 pRNA Three-Way Junction to Control Size and Shape of RNA Nanoparticles for Biodistribution Profiling in Mice.

RNA is rapidly emerging as a versatile building block for nanoparticle assembly due to its simplicity in base pairing, while exhibiting diversity in function such as enzymatic activity similar to some proteins. Recent advances in RNA nanotechnology have generated significant interests in applying RNA nanoparticles for various applications in nanotechnology and nanomedicine. In particular, assessing the effect of size and shape on cell entry and intracellular trafficking as well as in vivo biodistribution of nanoparticles is challenging due to the lack of nanoparticles rich in structure while varying in size and shape. RNA nanotechnology exemplified by the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor has provided a different prospect in nanoparticle designs. Of note, there is a robust three-way junction (3WJ) motif in pRNA which can serve as an adaptable scaffold to construct thermodynamically stable 2D planar and 3D globular RNA architectures with tunable shapes and sizes, and harboring various targeting, therapeutic, and imaging modules. This chapter focuses on the methods for constructing pRNA-3WJ based nanoparticles with controllable sizes and shapes, and assessment of their biodistribution profiles in cancer mouse models after systemic injection and ocular mouse models following subconjunctival injection.

Fingerprinting of Peptides with a Large Channel of Bacteriophage Phi29 DNA Packaging Motor.

Nanopore technology has become a highly sensitive and powerful tool for single molecule sensing of chemicals and biopolymers. Protein pores have the advantages of size amenability, channel homogeneity, and fabrication reproducibility. But most well-studied protein pores for sensing are too small for passage of peptide analytes that are typically a few nanometers in dimension. The funnel-shaped channel of bacteriophage phi29 DNA packaging motor has previously been inserted into a lipid membrane to serve as a larger pore with a narrowest N-terminal constriction of 3.6 nm and a wider C-terminal end of 6 nm. Here, the utility of phi29 motor channel for fingerprinting of various peptides using single molecule electrophysiological assays is reported. The translocation of peptides is proved unequivocally by single molecule fluorescence imaging. Current blockage percentage and distinctive current signatures are used to distinguish peptides with high confidence. Each peptide generated one or two distinct current blockage peaks, serving as typical fingerprint for each peptide. The oligomeric states of peptides can also be studied in real time at single molecule level. The results demonstrate the potential for further development of phi29 motor channel for detection of disease-associated peptide biomarkers.

Construction of RNA-Quantum Dot Chimera for Nanoscale Resistive Biomemory Application.

RNA nanotechnology offers advantages to construct thermally and chemically stable nanoparticles with well-defined shape and structure. Here we report the development of an RNA-QD (quantum dot) chimera for resistive biomolecular memory application. Each QD holds two copies of the pRNA three-way junction (pRNA-3WJ) of the bacteriophage phi29 DNA packaging motor. The fixed quantity of two RNAs per QD was achieved by immobilizing the pRNA-3WJ with a Sephadex aptamer for resin binding. Two thiolated pRNA-3WJ serve as two feet of the chimera that stand on the gold plate. The RNA nanostructure served as both an insulator and a mediator to provide defined distance between the QD and gold. Immobilization of the chimera nanoparticle was confirmed with scanning tunneling microscopy. As revealed by scanning tunneling spectroscopy, the conjugated pRNA-3WJ-QD chimera exhibited an excellent electrical bistability signal for biomolecular memory function, demonstrating great potential for the development of resistive biomolecular memory and a nano-bio-inspired electronic device for information processing and computing.

Single pore translocation of folded, double-stranded, and tetra-stranded DNA through channel of bacteriophage phi29 DNA packaging motor

The elegant architecture of the channel of bacteriophage phi29 DNA packaging motor has inspired the development of biomimetics for biophysical and nanobiomedical applications. The reengineered channel inserted into a lipid membrane exhibits robust electrophysiological properties ideal for precise sensing and fingerprinting of dsDNA at the single-molecule level. Herein, we used single channel conduction assays to quantitatively evaluate the translocation dynamics of dsDNA as a function of the length and conformation of dsDNA. We extracted the speed of dsDNA translocation from the dwell time distribution and estimated the various forces involved in the translocation process. A ∼35-fold slower speed of translocation per base-pair was observed for long dsDNA, a significant contrast to the speed of dsDNA crossing synthetic pores. It was found that the channel could translocate both dsDNA with ∼32% of channel current blockage and with ∼64% for tetra-stranded DNA (two parallel dsDNA). The calculation of both cross-sectional areas of the dsDNA and tetra-stranded DNA suggested that the blockage was purely proportional to the physical space of the channel lumen and the size of the DNA substrate. Folded dsDNA configuration was clearly reflected in their characteristic current signatures. The finding of translocation of tetra-stranded DNA with 64% blockage is in consent with the recently elucidated mechanism of viral DNA packaging via a revolution mode that requires a channel larger than the dsDNA diameter of 2 nm to provide room for viral DNA revolving without rotation. The understanding of the dynamics of dsDNA translocation in the phi29 system will enable us to design more sophisticated single pore DNA translocation devices for future applications in nanotechnology and personal medicine.

Using RNA nanoparticles with thermostable motifs and fluorogenic modules for real-time detection of RNA folding and turnover in prokaryotic and eukaryotic cells.

RNA nanotechnology is an emerging field at the interface of biochemistry and nanomaterials that shows immense promise for applications in nanomedicines, therapeutics and nanotechnology. Noncoding RNAs, such as siRNA, miRNA, ribozymes, and riboswitches, play important roles in the regulation of cellular processes. They carry out highly specific functions on a compact and efficient footprint. The properties of specificity and small size make them excellent modules in the construction of multifaceted RNA nanoparticles for targeted delivery and therapy. Biological activity of RNA molecules, however, relies on their proper folding. Therefore their thermodynamic and biochemical stability in the cellular environment is critical. Consequently, it is essential to assess global fold and intracellular lifetime of multifaceted RNA nanoparticles to optimize their therapeutic effectiveness. Here, we describe a method to express and assemble stable RNA nanoparticles in cells, and to assess the folding and turnover rate of RNA nanoparticles in vitro as well as in vivo in real time using a thermostable core motif derived from pRNA of bacteriophage Phi29 DNA packaging motor and fluorogenic RNA modules.

Functional assays for specific targeting and delivery of RNA nanoparticles to brain tumor.

Cumulative progress in nanoparticle development has opened a new era of targeted delivery of therapeutics to cancer cells and tissue. However, developing proper detection methods has lagged behind resulting in the lack of precise evaluation and monitoring of the systemically administered nanoparticles. RNA nanoparticles derived from the bacteriophage phi29 DNA packaging motor pRNA have emerged as a new generation of drugs for cancer therapy. Multifunctional RNA nanoparticles can be fabricated by bottom-up self-assembly of engineered RNA fragments harboring targeting (RNA aptamer or chemical ligand), therapeutic (siRNA, miRNA, ribozymes, and small molecule drugs), and imaging (fluorophore, radiolabels) modules. We have recently demonstrated that RNA nanoparticles can reach and target intracranial brain tumors in mice upon systemic injection with little or no accumulation in adjacent healthy brain tissues or in major healthy internal organs. Herein, we describe various functional imaging methods (fluorescence confocal microscopy, flow cytometry, fluorescence whole body imaging, and magnetic resonance imaging) to evaluate and monitor RNA nanoparticle targeting to intracranial brain tumors in mice. Such imaging techniques will allow in-depth evaluation of specifically delivered RNA therapeutics to brain tumors.

Robust Membrane-Embedded phi29 Motor Channel for Sensing of Single Molecule and High-Throughput Fingerprinting of DNA

The ingenious design of the bacteriophage phi29 DNA packaging motor with an elegant channel has inspired its applications in nanotechnology. The hub of the motor is a truncated cone shaped connector consisting of twelve protein subunits that form a ring with a central 3.6-nm channel that acts as a path for dsDNA to enter during packaging and to exit during infection. The connector inserted in lipid bilayer exhibits robust properties and generates extremely sensitive conductance signatures. DNA and RNA can be electrophoretically driven through the channel in a concentration and voltage dependent manner. Information about the structure, length and conformational dynamics can then be deduced by their characteristic dwell times during translocation and by their relative percentage in current blockades. This motor channel further exercises a one-way traffic property for dsDNA translocation from N- to C-terminal with a natural valve mechanism in DNA-packaging. We also demonstrated its utility as a highly sensitive device for capture and fingerprinting of chemicals and biopolymers in real time at extremely low concentrations and in the presence of many contaminants. The phi29 motor channel has potential applications in high-throughput single-pore DNA sequencing, environmental surveillance, athlete drug monitoring, toxin/drug screening, and earlier disease diagnosis.Supporting publications from Guo lab: (1) Nature Nanotechnology. 2009. 4: 765; (2) Nano Letters. 2010, 10: 3620; (3) Molecular Biosystems. 2010. 6:1844 (4) Biomaterials. 2011. 32:8234; (5) Biophysical Journal. 2012. 102:127; (6) ACS Nano. 2012. 6:3251; (7) Nature Protocols. 2013. 8:373; (8) Nano Today. 2013. 8:56; (9) ACS Nano. 2013. 7:9814.

Physicochemically tunable polyfunctionalized RNA square architecture with fluorogenic and ribozymatic properties.

Recent advances in RNA nanotechnology allow the rational design of various nanoarchitectures. Previous methods utilized conserved angles from natural RNA motifs to form geometries with specific sizes. However, the feasibility of producing RNA architecture with variable sizes using native motifs featuring fixed sizes and angles is limited. It would be advantageous to display RNA nanoparticles of diverse shape and size derived from a given primary sequence. Here, we report an approach to construct RNA nanoparticles with tunable size and stability. Multifunctional RNA squares with a 90° angle were constructed by tuning the 60° angle of the three-way junction (3WJ) motif from the packaging RNA (pRNA) of the bacteriophage phi29 DNA packaging motor. The physicochemical properties and size of the RNA square were also easily tuned by modulating the “core” strand and adjusting the length of the sides of the square via predictable design. Squares of 5, 10, and 20 nm were constructed, each showing diverse thermodynamic and chemical stabilities. Four “arms” extending from the corners of the square were used to incorporate siRNA, ribozyme, and fluorogenic RNA motifs. Unique intramolecular contact using the pre-existing intricacy of the 3WJ avoids relatively weaker intermolecular interactions via kissing loops or sticky ends. Utilizing the 3WJ motif, we have employed a modular design technique to construct variable-size RNA squares with controllable properties and functionalities for diverse and versatile applications with engineering, pharmaceutical, and medical potential. This technique for simple design to finely tune physicochemical properties adds a new angle to RNA nanotechnology.

Inhibition of monocyte adhesion to brain-derived endothelial cells by dual functional RNA chimeras.

Because adhesion of leukocytes to endothelial cells is the first step of vascular-neuronal inflammation, inhibition of adhesion and recruitment of leukocytes to vascular endothelial cells will have a beneficial effect on neuroinflammatory diseases. In this study, we used the pRNA of bacteriophage phi29 DNA packaging motor to construct a novel RNA nanoparticle for specific targeting to transferrin receptor (TfR) on the murine brain-derived endothelial cells (bEND5) to deliver ICAM-1 siRNA. This RNA nanoparticle (FRS-NPs) contained a FB4 aptamer targeting to TfR and a siRNA moiety for silencing the intercellular adhesion molecule-1 (ICAM-1). Our data indicated that this RNA nanoparticle was delivered into murine brain-derived endothelial cells. Furthermore, the siRNA was released from the FRS-NPs in the cells and knocked down ICAM-1 expression in the TNF-α–stimulated cells and in the cells under oxygen-glucose deprivation/reoxygenation (OGD/R) condition. The functional end points of the study indicated that FRS-NPs significantly inhibited monocyte adhesion to the bEND5 cells induced by TNF-α and OGD/R. In conclusion, our approach using RNA nanotechnology for siRNA delivery could be potentially applied for inhibition of inflammation in ischemic stroke and other neuroinflammatory diseases, or diseases affecting endothelium of vasculature.

Ocular Delivery of pRNA Nanoparticles: Distribution and Clearance After Subconjunctival Injection

RNA nanoparticles derived from the three-way junction (3WJ) of the pRNA of bacteriophage phi29 DNA packaging motor were previously found to be thermodynamically stable. As the nanoparticles could have potential in ocular drug delivery, the objectives in the present study were to investigate the distribution of pRNA nanoparticles after subconjunctival injection and examine the feasibility to deliver the nanoparticles to the cells of cornea and retina. Alexa647-labeled pRNA nanoparticles (pRNA-3WJ and pRNA-X) and double-stranded RNA (dsRNA) were administered via subconjunctival injection in mice. Alexa647 dye was a control. Topical administration was performed for comparison. Ocular clearance of pRNA nanoparticles and dsRNA after the injection was assessed using whole-body fluorescence imaging of the eyes. The numbers of cells in the ocular tissues with nanoparticle cell internalization were determined in fluorescence microscopy of dissected eye tissues. After subconjunctival injection, pRNA nanoparticles and dsRNA were observed to distribute into the eyes and cleared through the lymph. pRNA-3WJ, pRNA-X, and dsRNA were found in the cells of the conjunctiva, cornea, and sclera, but only pRNA-X was in the cells of the retina. Topical administration was not effective in delivering the nanoparticles to the eye. The pRNA nanoparticles were delivered to the cells in the eye via subconjunctival injection, and cell internalization was achieved in the cornea with pRNA-3WJ and pRNA-X and in the retina with pRNA-X. Only the X-shape pRNA-X could enter the retina.

Fabrication of pRNA nanoparticles to deliver therapeutic RNAs and bioactive compounds into tumor cells

RNA nanotechnology is a term that refers to the design, fabrication and use of nanoparticles that are mainly composed of RNAs via bottom-up self-assembly. The packaging RNA (pRNA) of the bacteriophage phi29 DNA packaging motor has been developed into a nanodelivery platform. This protocol describes the synthesis, assembly and functionalization of pRNA nanoparticles on the basis of three 'toolkits' derived from pRNA structural features: interlocking loops for hand-in-hand interactions, palindrome sequences for foot-to-foot interactions and an RNA three-way junction for branch extension. siRNAs, ribozymes, aptamers, chemical ligands, fluorophores and other functionalities can also be fused to the pRNA before the assembly of the nanoparticles, so as to ensure the production of homogeneous nanoparticles and the retention of appropriate folding and function of the incorporated modules. The resulting self-assembled multivalent pRNA nanoparticles are thermodynamically and chemically stable, and they remain intact at ultralow concentrations. Gene-silencing effects are progressively enhanced with increasing numbers of siRNAs in each pRNA nanoparticle. Systemic injection of the pRNA nanoparticles into xenograft-bearing mice has revealed strong binding to tumors without accumulation in vital organs or tissues. The pRNA-based nanodelivery scaffold paves a new way for nanotechnological application of pRNA-based nanoparticles for disease detection and treatment. The time required for completing one round of this protocol is 3-4 weeks when including in vitro functional assays, or 2-3 months when including in vivo studies.

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

The bacteriophage phi29 DNA packaging motor, one of the strongest biological motors characterized to date, is geared by a packaging RNA (pRNA) ring. When assembled from three RNA fragments, its three-way junction (3WJ) motif is highly thermostable, is resistant to 8 M urea, and remains associated at extremely low concentrations in vitro and in vivo. To elucidate the structural basis for its unusual stability, we solved the crystal structure of this pRNA 3WJ motif at 3.05 Å. The structure revealed two divalent metal ions that coordinate 4 nt of the RNA fragments. Single-molecule fluorescence resonance energy transfer (smFRET) analysis confirmed a structural change of 3WJ upon addition of Mg²⁺. The reported pRNA 3WJ conformation is different from a previously published construct that lacks the metal coordination sites. The phi29 DNA packaging motor contains a dodecameric connector at the vertex of the procapsid, with a central pore for DNA translocation. This portal connector serves as the foothold for pRNA binding to procapsid. Subsequent modeling of a connector/pRNA complex suggests that the pRNA of the phi29 DNA packaging motor exists as a hexameric complex serving as a sheath over the connector. The model of hexameric pRNA on the connector agrees with AFM images of the phi29 pRNA hexamer acquired in air and matches all distance parameters obtained from cross-linking, complementary modification, and chemical modification interference.

Revolution rather than rotation of AAA+ hexameric phi29 nanomotor for viral dsDNA packaging without coiling

It has long been believed that the DNA-packaging motor of dsDNA viruses utilizes a rotation mechanism. Here we report a revolution rather than rotation mechanism for the bacteriophage phi29 DNA packaging motor. The phi29 motor contains six copies of the ATPase (Schwartz et al., this issue); ATP binding to one ATPase subunit stimulates the ATPase to adopt a conformation with a high affinity for dsDNA. ATP hydrolysis induces a new conformation with a lower affinity, thus transferring the dsDNA to an adjacent subunit by a power stroke. DNA revolves unidirectionally along the hexameric channel wall of the ATPase, but neither the dsDNA nor the ATPase itself rotates along its own axis. One ATP is hydrolyzed in each transitional step, and six ATPs are consumed for one helical turn of 360°. Transition of the same dsDNA chain along the channel wall, but at a location 60° different from the last contact, urges dsDNA to move forward 1.75 base pairs each step (10.5bp per turn/6ATP=1.75bp per ATP). Each connector subunit tilts with a left-handed orientation at a 30° angle in relation to its vertical axis that runs anti-parallel to the right-handed dsDNA helix, facilitating the one-way traffic of dsDNA. The connector channel has been shown to cause four steps of transition due to four positively charged lysine rings that make direct contact with the negatively charged DNA phosphate backbone. Translocation of dsDNA into the procapsid by revolution avoids the difficulties during rotation that are associated with DNA supercoiling. Since the revolution mechanism can apply to any stoichiometry, this motor mechanism might reconcile the stoichiometry discrepancy in many phage systems where the ATPase has been found as a tetramer, hexamer, or nonamer.

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.