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A 24 x 30 nm ellipsoid nanoparticle containing 84 subunits or 7 dodecamers of the re-engineered core protein of the bacteriophage phi29 DNA packaging motor was constructed. Homogeneous nanoparticles were obtained with simple one-step purification. Electron microscopy and analytical ultracentrifugation were employed to elucidate the structure, shape, size, and mechanism of assembly. The formation of this structure was mediated and stabilized by N-terminal peptide extensions. Reversal of the 84-subunit ellipsoid nanoparticle to its dodecamer subunit was controlled by the cleavage of the extended N-terminal peptide with a protease. The 84 outward-oriented C-termini were conjugated with a streptavidin binding peptide which can be used for the incorporation of markers. This further extends the application of this nanoparticle to pathogen detection and disease diagnosis by signal enhancement.

Nanotubes are miniature materials with significant potential applications in nanotechnological, medical, biological and material sciences. The quest for manufacturing methods of nano-mechanical modules is in progress. For example, the application of carbon nanotubes has been extensively investigated due to the precise width control, but the precise length control remains challenging. Here we report two approaches for the one-pot self-assembly of RNA nanotubes. For the first approach, six RNA strands were used to assemble the nanotube by forming a 11 nm long hollow channel with the inner diameter of 1.7 nm and the outside diameter of 6.3 nm. For the second approach, six RNA strands were designed to hybridize with their neighboring strands by complementary base pairing and formed a nanotube with a six-helix hollow channel similar to the nanotube assembled by the first approach. The fabricated RNA nanotubes were characterized by gel electrophoresis and atomic force microscopy (AFM), confirming the formation of nanotube-shaped RNA nanostructures. Cholesterol molecules were introduced into RNA nanotubes to facilitate their incorporation into lipid bilayer. Incubation of RNA nanotube complex with the free-standing lipid bilayer membrane under applied voltage led to discrete current signatures. Addition of peptides into the sensing chamber revealed discrete steps of current blockage. Polyarginine peptides with different lengths can be detected by current signatures, suggesting that the RNA-cholesterol complex holds the promise of achieving single molecule sensing of peptides. [ABSTRACT FROM AUTHOR]

Aims: To find methods for potent drug development by targeting to biocomplex with high copy number. Methods: Phi29 DNA packaging motor components with different stoichiometries were used as model to assay virion assembly with Yang Hui's Triangle , where Z = stoichiometry, M = drugged subunits per biocomplex, p and q are the fraction of drugged and undrugged subunits in the population. Results: Inhibition efficiency follows a power function. When number of drugged subunits to block the function of the complex K = 1, the uninhibited biocomplex equals qz, demonstrating the multiplicative effect of stoichiometry on inhibition with stoichiometry 1000 > 6 > 1. Complete inhibition of virus replication was found when Z = 6. Conclusion: Drug inhibition potency depends on the stoichiometry of the targeted components of the biocomplex or nanomachine. The inhibition effect follows a power function of the stoichiometry of the target biocomplex. [ABSTRACT FROM AUTHOR]

Living systems produce ordered structures and nanomachines that inspire the development of biomimetic nanodevices such as chips, MEMS, actuators, sensors, sorters, and apparatuses for single-pore DNA sequencing, disease diagnosis, drug or therapeutic RNA delivery. Determination of the copy numbers of subunits that build these machines is challenging due to small size. Here we report a simple mathematical method to determine the stoichiometry, using phi29 DNA-packaging nanomotor as a model to elucidate the application of a formula ∑ M = 0 Z Z M p Z − M q M , where p and q are the percentage of wild-type and inactive mutant in the empirical assay; M is the copy numbers of mutant and Z is the stoichiometry in question. Variable ratios of mutants and wild-type were mixed to inhibit motor function. Empirical data were plotted over the theoretical curves to determine the stoichiometry and the value of K, which is the number of mutant needed in each machine to block the function, all based on the condition that wild-type and mutant are equal in binding affinity. Both Z and K from 1-12 were investigated. The data precisely confirmed that phi29 motor contains six copies (Z) of the motor ATPase gp16, and K = 1. From the Clinical Editor To determine copy numbers of subunits that form nanomachines in living organisms is a daunting task due to the complexities and the inherently small sizes associated with such systems. In this paper, a simple mathematical method is described how to determine the stoichiometry of copies in biomimetic nanodevices, using phi29 DNA-packaging nanomotor as a model. [ABSTRACT FROM AUTHOR]

Abstract: Biomotors have been classified into linear and rotational motors. For 35 years, it has been popularly believed that viral dsDNA-packaging apparatuses are pentameric rotation motors. Recently, a third class of hexameric motor has been found in bacteriophage phi29 that utilizes a mechanism of revolution without rotation, friction, coiling, or torque. This review addresses how packaging motors control dsDNA one-way traffic; how four electropositive layers in the channel interact with the electronegative phosphate backbone to generate four steps in translocating one dsDNA helix; how motors resolve the mismatch between 10.5 bases and 12 connector subunits per cycle of revolution; and how ATP regulates sequential action of motor ATPase. Since motors with all number of subunits can utilize the revolution mechanism, this finding helps resolve puzzles and debates concerning the oligomeric nature of packaging motors in many phage systems. This revolution mechanism helps to solve the undesirable dsDNA supercoiling issue involved in rotation. [Copyright &y& Elsevier]

Summary: Sensitivity and specificity are two most important factors to take into account for molecule sensing, chemical detection and disease diagnosis. A perfect sensitivity is to reach the level where a single molecule can be detected. An ideal specificity is to reach the level where the substance can be detected in the presence of many contaminants. The rapidly progressing nanopore technology is approaching this threshold. A wide assortment of biomotors and cellular pores in living organisms perform diverse biological functions. The elegant design of these transportation machineries has inspired the development of single molecule detection based on modulations of the individual current blockage events. The dynamic growth of nanotechnology and nanobiotechnology has stimulated rapid advances in the study of nanopore based instrumentation over the last decade, and inspired great interest in sensing of single molecules including ions, nucleotides, enantiomers, drugs, and polymers such as PEG, RNA, DNA, and polypeptides. This sensing technology has been extended to medical diagnostics and third generation high throughput DNA sequencing. This review covers current nanopore detection platforms including both biological pores and solid state counterparts. Several biological nanopores have been studied over the years, but this review will focus on the three best characterized systems including α-hemolysin and MspA, both containing a smaller channel for the detection of single stranded DNA, as well as bacteriophage phi29 DNA packaging motor connector that contains a larger channel for the passing of double stranded DNA. The advantage and disadvantage of each system are compared; their current and potential applications in nanomedicine, biotechnology, and nanotechnology are discussed. [Copyright &y& Elsevier]

Summary: One of the advantages of nanotechnology is the feasibility to construct therapeutic particles carrying multiple therapeutics with defined structure and stoichiometry. The field of RNA nanotechnology is emerging. However, controlled assembly of stable RNA nanoparticles with multiple functionalities which retain their original role is challenging due to refolding after fusion. Herein, we report the construction of thermodynamically stable X-shaped RNA nanoparticles to carry four therapeutic RNA motifs by self-assembly of reengineered small RNA fragments. We proved that each arm of the four helices in the X-motif can harbor one siRNA, ribozyme, or aptamer without affecting the folding of the central pRNA-X core, and each daughter RNA molecule within the nanoparticle folds into their respective authentic structures and retains their biological and structural function independently. Gene silencing effects were progressively enhanced as the number of the siRNA in each pRNA-X nanoparticles gradually increased from one to two, three, and four. More importantly, systemic injection of ligand-containing nanoparticles into the tail-vein of mice revealed that the RNA nanoparticles remained intact and strongly bound to cancers without entering the liver, lung or any other organs or tissues, while remaining in cancer tissue for more than 8h. [Copyright &y& Elsevier]

Abstract: RNA engineering for nanotechnology and medical applications is an exciting emerging research field. RNA has intrinsically defined features on the nanometre 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 downregulate gene expression in a temporally- and spatially controlled fashion has led to heightened interest and rapid development of siRNA-based therapeutics. Although methods for gene silencing have been achieved with high efficacy and specificity in vitro, the effective delivery of nucleic acids to specific cells in vivo has been a hurdle for RNA therapeutics. This article covers different RNA-based approaches for diagnosis, prevention and treatment of human disease, with a focus on the latest developments of non-viral 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. [Copyright &y& Elsevier]

The past decade has shown exponential growth in the field of RNA nanotechnology. The rapid advances of using RNA nanoparticles for biomedical applications, especially targeted cancer therapy, suggest its potential as a new generation of drug. After the first milestone of small molecule drugs and the second milestone of antibody drugs, it was predicted that RNA drugs, either RNA itself or chemicals/ligands that target RNA, will be the third milestone in drug development. Thus, a comprehensive assessment of the current therapeutic RNA nanoparticles is urgently needed to meet the drug evaluation criteria. Specifically, the pharmacological and immunological profiles of RNA nanoparticles need to be systematically studied to provide insights in rational design of RNA‐based therapeutics. By virtue of its programmability and biocompatibility, RNA molecules can be designed to construct sophisticated nanoparticles with versatile functions/applications and highly tunable physicochemical properties. This intrinsic characteristic allows the systemic study of the effects of various properties of RNA nanoparticles on their in vivo behaviors such as cancer targeting and immune responses. This review will focus on the recent progress of RNA nanoparticles in cancer targeting, and summarize the effects of common physicochemical properties such as size and shape on the RNA nanoparticles' biodistribution and immunostimulation profiles. This article is categorized under: Biology‐Inspired Nanomaterials > Nucleic Acid‐Based Structures Diagnostic Tools > in vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease [ABSTRACT FROM AUTHOR]

Abstract: The potent ability of small interfering RNA (siRNA) to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for diseases including HIV. However, efficient delivery of siRNAs remains a key obstacle to successful application. A targeted intracellular delivery approach for siRNAs to specific cell types is highly desirable. HIV-1 infection is initiated by the interactions between viral glycoprotein gp120 and cell surface receptor CD4, leading to fusion of the viral membrane with the target cell membrane. Once HIV infects a cell it produces gp120 which is displayed at the cell surface. We previously described a novel dual inhibitory anti-gp120 aptamer–siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. We also demonstrated that gp120 can be used for aptamer mediated delivery of anti-HIV siRNAs. Here we report the design, construction and evaluation of chimerical RNA nanoparticles containing a HIV gp120-binding aptamer escorted by the pRNA of bacteriophage phi29 DNA-packaging motor. We demonstrate that pRNA–aptamer chimeras specifically bind to and are internalized into cells expressing HIV gp120. Moreover, the pRNA–aptamer chimeras alone also provide HIV inhibitory function by blocking viral infectivity. The Ab′ pRNA–siRNA chimera with 2′-F modified pyrimidines in the sense strand not only improved the RNA stability in serum, but also was functionally processed by Dicer, resulting in specific target gene silencing. Therefore, this dual functional pRNA–aptamer not only represents a potential HIV-1 inhibitor, but also provides a cell-type specific siRNA delivery vehicle, showing promise for systemic anti-HIV therapy. [Copyright &y& Elsevier]

Liver or other organ accumulation of drugs is one of the major problems that leads to toxicity and side effects in therapy using chemicals or other macromolecules. It has been shown that specially designed RNA nanoparticles can specifically target cancer cells, silence oncogenic genes, and stop cancer growth with little or no accumulation in the liver or other vital organs. It is well known that physical properties of nanoparticles such as size, shape, and surface chemistry affect biodistribution and pharmacokinetic profiles in vivo. This study examined how the hydrophobicity of chemicals conjugated to RNA nanoparticles affect in vivo biodistribution. Weaker organ accumulation was observed for hydrophobic chemicals after they were conjugated to RNA nanoparticles, revealing RNA's ability to solubilize hydrophobic chemicals. It was found that different chemicals conjugated to RNA nanoparticles resulted in the alteration of RNA hydrophobicity. Stronger hydrophobicity induced by chemical conjugates resulted in higher accumulation of RNA nanoparticles in vital organs in mice. This study provides new insights for handling drug insolubility, therapeutic toxicity, and organ clearance in drug development. [ABSTRACT FROM AUTHOR]

Nanopore technology has become a powerful tool in single molecule sensing, and protein nanopores appear to be more advantageous than synthetic counterparts with regards to channel amenability, structure homogeneity, and production reproducibility. However, the diameter of most of the well-studied protein nanopores is too small to allow the passage of protein or peptides that are typically in multiple nanometers scale. The portal channel from bacteriophage SPP1 has a large channel size that allows the translocation of peptides with higher ordered structures. Utilizing single channel conductance assay and optical single molecule imaging, we observed translocation of peptides and quantitatively analyzed the dynamics of peptide oligomeric states in real-time at single molecule level. The oxidative and the reduced states of peptides were clearly differentiated based on their characteristic electronic signatures. A similar Gibbs free energy (ΔG 0 ) was obtained when different concentrations of substrates were applied, suggesting that the use of SPP1 nanopore for real-time quantification of peptide oligomeric states is feasible. With the intrinsic nature of size and conjugation amenability, the SPP1 nanopore has the potential for development into a tool for the quantification of peptide and protein structures in real time. [ABSTRACT FROM AUTHOR]

Insertion of biological nanopore into artificial membrane is of fundamental importance in nanotechnology. Many applications require control and knowledge of channel orientation. In this work, the insertion orientation of the bacteriophage SPP1 and phi29 DNA packaging motors into lipid membranes was investigated. Single molecule electrophysiological assays and Ni-NTA-nanogold binding assays revealed that both SPP1 and phi29 motor channels exhibited a one-way traffic property for TAT peptide translocation from N- to C-termini of the protein channels. SPP1 motor channels preferentially inserts into liposomes with their C-terminal wider region facing inward. Changing the hydrophobicity of the N- or C-termini of phi29 connector alters the insertion orientation, suggesting that the hydrophobicity and hydrophilicity of the termini of the protein channel governs the orientation of the insertion into lipid membrane. It is proposed that the specificity in motor channel orientation is a result of the hydrophilic/hydrophobic interaction at the air/water interface when the protein channels are incorporating into liposome membranes. [ABSTRACT FROM AUTHOR]

In optical devices such as camera or microscope, an aperture is used to regulate light intensity for imaging. Here we report the discovery and construction of a durable bio-aperture at nanometerscale that can regulate current at the pico-ampere scale. The nano-aperture is made of 12 identical protein subunits that form a 3.6-nm channel with a shutter and "one-way traffic" property. This shutter responds to electrical potential differences across the aperture and can be turned off for double stranded DNA translocation. This voltage enables directional control, and three-step regulation for opening and closing. The nano-aperture was constructed in vitro and purified into homogeneity. The aperture was stable at pH2-12, and a temperature of −85C–60C. When an electrical potential was held, three reproducible discrete steps of current flowing through the channel were recorded. Each step reduced 32% of the channel dimension evident by the reduction of the measured current flowing through the aperture. The current change is due to the change of the resistance of aperture size. The transition between these three distinct steps and the direction of the current was controlled via the polarity of the voltage applied across the aperture. When the C-terminal of the aperture was fused to an antigen, the antibody and antigen interaction resulted in a 32% reduction of the channel size. This phenomenon was used for disease diagnosis since the incubation of the antigen-nano-aperture with a specific cancer antibody resulted in a change of 32% of current. The purified truncated cone-shape aperture automatically self-assembled efficiently into a sheet of the tetragonal array via head-to-tail self-interaction. The nano-aperture discovery with a controllable shutter, discrete-step current regulation, formation of tetragonal sheet, and one-way current traffic provides a nanoscale electrical circuit rectifier for nanodevices and disease diagnosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Human genome sequencing revealed that only ~1.5% of the DNA sequence coded for proteins. More and more evidence has uncovered that a substantial part of the 98.5% so‐called “junk” DNAs actually code for noncoding RNAs. Two milestones, chemical drugs and protein drugs, have already appeared in the history of drug development, and it is expected that the third milestone in drug development will be RNA drugs or drugs that target RNA. This review focuses on the development of RNA therapeutics for potential cancer treatment by applying RNA nanotechnology. A therapeutic RNA nanoparticle is unique in that its scaffold, ligand, and therapeutic component can all be composed of RNA. The special physicochemical properties lend to the delivery of siRNA, miRNA, ribozymes, or riboswitches; imaging using fluogenenic RNA; and targeting using RNA aptamers. With recent advances in solving the chemical, enzymatic, and thermodynamic stability issues, RNA nanoparticles have been found to be advantageous for in vivo applications due to their uniform nano-scale size, precise stoichiometry, polyvalent nature, low immunogenicity, low toxicity, and target specificity. In vivo animal studies have revealed that RNA nanoparticles can specifically target tumors with favorable pharmacokinetic and pharmacodynamic parameters without unwanted accumulation in normal organs. This review summarizes the key studies that have led to the detailed understanding of RNA nanoparticle formation as well as chemical and thermodynamic stability issue. The methods for RNA nanoparticle construction, and the current challenges in the clinical application of RNA nanotechnology, such as endosome trapping and production costs, are also discussed. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: The AAA+ superfamily of proteins is a class of motor ATPases performing a wide range of functions that typically exist as hexamers. The ATPase of phi29 DNA packaging motor has long been a subject of debate in terms of stoichiometry and mechanism of action. Here, we confirmed the stoichiometry of phi29 motor ATPase to be a hexamer and provide data suggesting that the phi29 motor ATPase is a member of the classical hexameric AAA+ superfamily. Native PAGE, EMSA, capillary electrophoresis, ATP titration, and binomial distribution assay show that the ATPase is a hexamer. Mutations in the known Walker motifs of the ATPase validated our previous assumptions that the protein exists as another member of this AAA+ superfamily. Our data also supports the finding that the phi29 DNA packaging motor uses a revolution mechanism without rotation or coiling (Schwartz et al., this issue). [Copyright &y& Elsevier]

Abstract: Recent advances in RNA nanotechnology have led to the emergence of a new field and brought vitality to the area of therapeutics [P. Guo, The emerging field of RNA nanotechnology, Nat. Nanotechnol., 2010]. Due to the complementary nature of the four nucleotides and its special catalytic activity, RNA can be manipulated with simplicity characteristic of DNA, while possessing versatile structure and diverse function similar to proteins. Loops and tertiary architecture serve as mounting dovetails or wedges to eliminate external linking dowels. Unique features in transcription, termination, self-assembly, self-processing, and acid-resistance enable in vivo production of nanoparticles harboring aptamer, siRNA, ribozyme, riboswitch, or other regulators for therapy, detection, regulation, and intracellular computation. The unique property of noncanonical base-pairing and stacking enables RNA to fold into well-defined structures for constructing nanoparticles with special functionalities. Bacteriophage phi29 DNA packaging motor is geared by a ring consisting of six packaging RNA (pRNA) molecules. pRNA is able to form a multimeric complex via the interaction of two reengineered interlocking loops. This unique feature makes it an ideal polyvalent vehicle for nanomachine fabrication, pathogen detection, and delivery of siRNA or other therapeutics. This review describes methods in using pRNA as a building block for the construction of RNA dimers, trimers, and hexamers as nanoparticles in medical applications. Methods for industrial-scale production of large and stable RNA nanoparticles will be introduced. The unique favorable PK (pharmacokinetics) profile with a half life (T 1/2) of 5–10h comparing to 0.25 of conventional 2′-F siRNA, and advantageous in vivo features such as non-toxicity, non-induction of interferons or non-stimulating of cytokine response in animals will also be reviewed. [Copyright &y& Elsevier]