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Fabrication of Massive Sheets of Single Layer Patterned Arrays Using Lipid Directed Reengineered Phi29 Motor Dodecamer
Abstract
The bottom-up assembly of patterned arrays is an exciting and important area in current nanotechnology. Arrays can be engineered to serve as components in chips for a virtually inexhaustible list of applications ranging from disease diagnosis to ultra-high-density data storage. Phi29 motor dodecamer has been reported to form elegant multilayer tetragonal arrays. However, multilayer protein arrays are of limited use for nanotechnological applications which demand nanoreplica or coating technologies. The ability to produce a single layer array of biological structures with high replication fidelity represents a significant advance in the area of nanomimetics. In this paper, we report on the assembly of single layer sheets of reengineered phi29 motor dodecamer. A thin lipid monolayer was used to direct the assembly of massive sheets of single layer patterned arrays of the reengineered motor dodecamer. Uniform, clean and highly ordered arrays were constructed as shown by both transmission electron microscopy and atomic force microscopy imaging.
... [10][11][12][13] Explicit protein engineering via site-directed mutagenesis of the phi29 connector is possible because the crystal structure of the wild-type connector has been elucidated. [14][15][16] It is therefore feasible to reengineer the connector by introducing specific binding elements at strategic positions (at the C-or N-termini or in the interior of the channel) for desired applications [17][18][19] . The phi29 connector consists of 12 subunits of the gp10 protein that form a central channel shaped like a hollow truncated cone. ...
... 25 The procedures for large-scale production and purification of the connector have also been developed. 17,[26][27][28][29] This protocol focuses on the cloning, expression and purification of the reengineered connector protein and its insertion into a lipid membrane setup, thereby creating a robust lipid bilayer-embedded nanopore platform that is capable of single-molecule detection of biopolymers and chemicals. ...
Over the past decade, nanopores have rapidly emerged as stochastic biosensors. This protocol describes the cloning, expression and purification of the channel of the bacteriophage phi29 DNA-packaging nanomotor and its subsequent incorporation into lipid membranes for single-pore sensing of double-stranded DNA (dsDNA) and chemicals. The membrane-embedded phi29 nanochannel remains functional and structurally intact under a range of conditions. When ions and macromolecules translocate through this nanochannel, reliable fingerprint changes in conductance are observed. Compared with other well-studied biological pores, the phi29 nanochannel has a larger cross-sectional area, which enables the translocation of dsDNA. Furthermore, specific amino acids can be introduced by site-directed mutagenesis within the large cavity of the channel to conjugate receptors that are able to bind specific ligands or analytes for desired applications. The lipid membrane-embedded nanochannel system has immense potential nanotechnological and biomedical applications in bioreactors, environmental sensing, drug monitoring, controlled drug delivery, early disease diagnosis and high-throughput DNA sequencing. The total time required for completing one round of this protocol is around 1 month.
... In the phi29 DNA packaging motor, the pRNA forms dimers as the building blocks for the hexameric conformation. Six phi29 pRNAs can assemble into a hexameric pRNA ring through intermolecular base-pairing, which can be observed by transmission electron microscopy with nanogold-labeled pRNA [52][53][54] and this phi29 DNA packaging motor represents one of the strongest biomotors studied to date. ...
Background:
This attractive and intriguing Ribonucleic acid (RNA) nanotechnology has been conceptualized over the last two decades and with our increasing understanding of RNA structure and function and improvements of RNA nanotechnology it is now possible to use this in clinical settings.
Methods:
Here we review the unique properties and the recent advances in RNA nanotechnology and then look at its scientific and preclinical applications for tumor diagnosis and targeted delivery and RNA-based therapy using RNA nanoparticles with diverse structures and functions. Finally, we discuss the future perspectives and challenges to RNA nanotechnology.
Results:
RNA can be designed and manipulated in a similar way to DNA while having different rules for base-pairing and displaying functions similar to proteins. Rationally designed RNA nanoparticles based on the three-way junction (3WJ) motif as the core scaffold have been extensively explored in the field of nanomedicine and targeted cancer diagnosis and therapy.
Conclusions:
RNA nanostructures based on 3WJs demonstrate promising future applications due to their thermal stability, molecular-level plasticity, multifunctional chemotherapeutic drug delivery and other intrinsic characteristics, which will greatly improve the treatment of cancer and promote further major breakthroughs in this field.
... Despite their functional diversity, a common feature of the motors of this family is their ability to convert energy obtained from the binding or hydrolysis of ATP into mechanical energy which results in local/global protein unfolding, complex assembly/disassembly, or grabbing/ pushing dsDNA for translocation [1][2][3][4][5][6][7][8][9][10][11]. The hexagonal shape of the motor facilitates bottom-up assembly in nanomachine manufacturing [12][13][14][15][16][17][18][19][20]. ...
Background
Double-stranded DNA translocation is ubiquitous in living systems. Cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging and cell entry all involve biomotor-driven dsDNA translocation. Previously, biomotors have been primarily classified into linear and rotational motors. We recently discovered a third class of dsDNA translocation motors in Phi29 utilizing revolution mechanism without rotation. Analogically, the Earth rotates around its own axis every 24 hours, but revolves around the Sun every 365 days.
Results
Single-channel DNA translocation conductance assay combined with structure inspections of motor channels on bacteriophages P22, SPP1, HK97, T7, T4, Phi29, and other dsDNA translocation motors such as bacterial FtsK and eukaryotic mimiviruses or vaccinia viruses showed that revolution motor is widespread. The force generation mechanism for revolution motors is elucidated. Revolution motors can be differentiated from rotation motors by their channel size and chirality. Crystal structure inspection revealed that revolution motors commonly exhibit channel diameters larger than 3 nm, while rotation motors that rotate around one of the two separated DNA strands feature a diameter smaller than 2 nm. Phi29 revolution motor translocated double- and tetra-stranded DNA that occupied 32% and 64% of the narrowest channel cross-section, respectively, evidencing that revolution motors exhibit channel diameters significantly wider than the dsDNA. Left-handed oriented channels found in revolution motors drive the right-handed dsDNA via anti-chiral interaction, while right-handed channels observed in rotation motors drive the right-handed dsDNA via parallel threads. Tethering both the motor and the dsDNA distal-end of the revolution motor does not block DNA packaging, indicating that no rotation is required for motors of dsDNA phages, while a small-angle left-handed twist of dsDNA that is aligned with the channel could occur due to the conformational change of the phage motor channels from a left-handed configuration for DNA entry to a right-handed configuration for DNA ejection for host cell infection.
Conclusions
The revolution motor is widespread among biological systems, and can be distinguished from rotation motors by channel size and chirality. The revolution mechanism renders dsDNA void of coiling and torque during translocation of the lengthy helical chromosome, thus resulting in more efficient motor energy conversion.
... predictable self-assembly patterns represents a valuable tool for nanopatterning. Efforts to use proteins as patterning elements included engineering a protein of the viral DNApackaging machinery [13] and making fusion proteins that self-assembled into large, symmetrical nanostructures [14]. ...
The ability of isolated bacterial cell surface layer (S-layer) protein subunits to self-assemble into protein layers on solid surfaces makes them an almost ideal biological template for sensor oriented applications. Here, we used this remarkable property of the recombinant S-layer protein of Sporosarcina ureae bacterium to display streptavidin in defined order and orientation into two dimensional, nanosized protein lattices. We show that these lattices constitute a functional biomolecular matrix by binding biotinylated quantum dots on its surface. Therefore, they have a great application potential as sensor elements.
... One of the two approaches in nanotechnology is bottom-up assembly that uses modified or engineered building blocks (Schmidt and Eberl 2001; Balzani et al. 2002; Seeman and Belcher 2002; Shu et al. 2004; Xiao et al. 2009a,b, 2010). RNA is versatile in structure and function and even possesses enzymatic activity characteristic of some proteins (Nilsen 2007; Guo 2010; Shukla et al. 2011). ...
Due to structural flexibility, RNase sensitivity, and serum instability, RNA nanoparticles with concrete shapes for in vivo application remain challenging to construct. Here we report the construction of 14 RNA nanoparticles with solid shapes for targeting cancers specifically. These RNA nanoparticles were resistant to RNase degradation, stable in serum for >36 h, and stable in vivo after systemic injection. By applying RNA nanotechnology and exemplifying with these 14 RNA nanoparticles, we have established the technology and developed "toolkits" utilizing a variety of principles to construct RNA architectures with diverse shapes and angles. The structure elements of phi29 motor pRNA were utilized for fabrication of dimers, twins, trimers, triplets, tetramers, quadruplets, pentamers, hexamers, heptamers, and other higher-order oligomers, as well as branched diverse architectures via hand-in-hand, foot-to-foot, and arm-on-arm interactions. These novel RNA nanostructures harbor resourceful functionalities for numerous applications in nanotechnology and medicine. It was found that all incorporated functional modules, such as siRNA, ribozymes, aptamers, and other functionalities, folded correctly and functioned independently within the nanoparticles. The incorporation of all functionalities was achieved prior, but not subsequent, to the assembly of the RNA nanoparticles, thus ensuring the production of homogeneous therapeutic nanoparticles. More importantly, upon systemic injection, these RNA nanoparticles targeted cancer exclusively in vivo without accumulation in normal organs and tissues. These findings open a new territory for cancer targeting and treatment. The versatility and diversity in structure and function derived from one biological RNA molecule implies immense potential concealed within the RNA nanotechnology field.
... 32,33 This dodecameric connector protein has shown great potential in nanotechnology and nanomedicine applications because of its ability to form peptide-mediated, 34,35 as well as nucleotidemediated, 36 self-assembled nanoparticles. Also advantageous is the realization that it can be constructed into multilayers 37,38 and single-layer patterned arrays, 39 and it has a high sensitivity for real-time sensing of nucleotides and single chemicals. 40,41 In 1978, an attractive model with a five-fold/six-fold rotation mechanism for bacteriophage dsDNA packaging was proposed. ...
The importance of nanomotors in nanotechnology is akin to that of mechanical engines to daily life. The AAA+ superfamily is a class of nanomotors performing various functions. Their hexagonal arrangement facilitates bottom-up assembly for stable structures. Bacteriophage phi29 DNA-translocation motor contains three co-axial rings: a dodecamer channel, a hexameric ATPase ring, and a hexameric pRNA ring. Viral DNA-packaging motor has been believed to be a rotational machine. However, we discovered a revolution mechanism without rotation. By analogy, the earth revolves around the sun while rotating on its own axis. One-way traffic of dsDNA translocation is facilitated by five factors: 1) ATPase changes its conformation to revolve dsDNA within hexameric channel in one direction; 2) the 30° tilt of the channel subunits causes an anti-parallel arrangement between two helices of dsDNA and channel wall to advance one-way translocation; 3) unidirectional flow property of the internal channel loops serves as a ratchet valve to prevent reversal; 4) 5'-3' single-direction movement of one DNA strand along the channel wall ensures single direction; and 5) four electropositive layers interact with one strand of the electronegative dsDNA phosphate backbone, resulting in four relaying transitional pauses during translocation. The discovery of a riding system along one strand provides a motion nano-system for cargo transportation and a tool for studying force generation without coiling, friction, and torque. The revolution of dsDNA among 12 subunits offers a series of recognition sites on DNA backbone to provide additional spatial variables for nucleotides discrimination for sensing applications.
... 9,10 The pore size of the connector is nearly identical from sample to sample and chemical conjugations within the large cavity of the pore can be made for added functionality with relative ease 11 . Furthermore, the procedures for large scale production and purification of the connector have already been developed, 10,[12][13][14][15][16] and anchoring within the viral capsid is mediated via protein-protein interactions. ...
Nanopores have been utilized to detect the conformation and dynamics of polymers, including DNA and RNA. Biological pores are extremely reproducible at the atomic level with uniform channel sizes. The channel of the bacterial virus phi29 DNA packaging motor is a natural conduit for the transportation of double-stranded DNA (dsDNA), and has the largest diameter among the well-studied biological channels. The larger channel facilitates translocation of dsDNA, and offers more space for further channel modification and conjugation. Interestingly, the relatively large wild type channel, which translocates dsDNA, cannot detect single-stranded nucleic acids (ssDNA or ssRNA) under the current experimental conditions. Herein, we reengineered this motor channel by removing the internal loop segment of the channel. The modification resulted in two classes of channels. One class was the same size as the wild type channel, while the other class had a cross-sectional area about 60% of the wild type. This smaller channel was able to detect the real-time translocation of single stranded nucleic acids at single-molecule level. While the wild type connector exhibited a one-way traffic property with respect to dsDNA translocation, the loop deleted connector was able to translocate ssDNA and ssRNA with equal competencies from both termini. This finding of size alterations in reengineered motor channels expands the potential application of the phi29 DNA packaging motor in nanomedicine, nanobiotechnology, and high-throughput single pore DNA sequencing.
... Biological materials, in the form of DNA, RNA, protein, and lipids, serve as models for the self-recognition and self-assembly of bionanoparticles. 13 Peptides also play a unique role in nanostructure design, owing to their diversity, simplicity of synthesis, and ease of modifying them for a variety of functions. Understanding the self-assembly mechanism of these biomaterials enables us to design and engineer biomimetics on a nanoscale. ...
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.
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.
Bacteriophage phi29 DNA packaging motor consists of a dodecameric portal channel protein complex termed connector that allows transportation of genomic dsDNA and a hexameric packaging RNA (pRNA) ring to gear the motor. The elegant design of the portal protein has facilitated its applications for real-time single-molecule detection of biopolymers and chemicals with high sensitivity and selectivity. The robust self-assembly property of the pRNA has enabled biophysical studies of the motor complex to determine the stoichiometry and structure/folding of the pRNA at single-molecule level. This chapter focuses on biophysical and analytical methods for studying the phi29 motor components at the single-molecule level, such as single channel conductance assays of membrane-embedded connectors; single molecule photobleaching (SMPB) assay for determining the stoichiometry of phi29 motor components; fluorescence resonance energy transfer (FRET) assay for determining the structure and folding of pRNA; atomic force microscopy (AFM) for imaging pRNA nanoparticles of various size, shape, and stoichiometry; and bright-field microscopy with magnetomechanical system for direct visualization of viral DNA packaging process. The phi29 system with explicit engineering capability has incredible potentials for diverse applications in nanotechnology and nanomedicine including, but not limited to, DNA sequencing, drug delivery to diseased cells, environmental surveillance, and early disease diagnosis.
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.
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.
Copyright © 2015 Elsevier Ltd. All rights reserved.
The ingenious design of the bacterial virus phi29 DNA packaging nano-motor with an elegant and elaborate channel has inspired its application for single molecule detection of antigen/antibody interactions. The hub of this bacterial virus nanomotor is a truncated cone-shaped connector consisting of twelve protein subunits. These subunits form a ring with a central 3.6-nm channel acting as a path for dsDNA to enter during packaging and to exit during infection. The connector has been inserted into a lipid bilayer. Herein, we reengineered an Epithelial Cell Adhesion Molecule (EpCAM) peptide into the C-terminal of nanopore as a probe to specifically detect EpCAM antibody (Ab) in nano-molar concentration at the single molecule level. The binding of Abs sequentially to each peptide probe induced step-wise blocks in current. The distinctive current signatures enabled us to analyze the docking and undocking kinetics of Ab-probe interactions and determine the Kd. The signal of EpCAM antibody can be discriminated from the background events in the presence of non-specific antibody or serum. Our results demonstrate the feasibility of generating a highly sensitive platform for detecting antibodies at extremely low concentrations in the presence of contaminants.
Nanobiotechnology entails the use of nanosized materials to build structures that can be applied in both biotechnology and medicine. In one vein of this field of study, scientists seek to mimic the wide variety of nanomachines and macromolecular structures that exist in nature and to replicate them in both structure and function. As a most intriguing example, the bacterial virus phi29 uses a self-contained nanomotor to package its DNA after replication. The 30-nm nanomotor contains 12 copies of a protein (gp10) which together form a 3.6-nm central channel through which the genomic DNA passes into the procapsid during viral assembly and exits during infection. This connector has been recently reengineered and embedded into a lipid bilayer, creating a system with tremendous application for DNA detection and characterization through electrophysiological measurement. A second component of the phi29 bacteriophage is an ATP-binding pRNA that forms a hexameric ring to gear the motor. The pRNA has been utilized to construct nanoparticles of dimers, trimers, hexamers and patterned superstructures via the interaction of two interlocking loops. Such structures constructed via bottom-up assembly have been used in the delivery of drugs, siRNA, ribozymes, and genes to specific cells, both in vitro and in vivo. This review summarizes current studies on the structure, function, and mechanism of the phi29 DNA packaging motor, as well as addresses the applications of these motor components in the field of nanobiotechnology.
An efficient method to form lipid bilayers inside an array of microfluidic channels has been developed and applied to monitor the membrane-embedded phi29 DNA packaging motor with an electrochemical characterization on a lab-on-a-chip (LOC) platform. A push-pull junction capturing approach was applied to confine a small amount of the lipid solution inside a microchannel. The selective permeability between solvents and water in PDMS was utilized to extract the solvent from the lipid solution, resulting in a self-formation of the lipid bilayer in the microchannel array. Each microchannel was independently connected to a silver/silver chloride (Ag/AgCl) electrode array, leading to a high-throughput monitoring of the nanopore insertion in the formed lipid bilayers. The formation of multiple lipid bilayers inside an array of microchannels and the simultaneous electrical and optical monitoring of multiple bilayer provides an efficient LOC platform for the further development of single phi29 motor pore sensing and high throughput single pore dsDNA sequencing.
Double-stranded (ds)DNA viruses package their genomic DNA into a procapsid using a force-generating nanomotor powered by ATP hydrolysis. Viral DNA packaging motors are mainly composed of the connector channel and two DNA packaging enzymes. In 1998, it was proposed that viral DNA packaging motors exercise a mechanism similar to the action of AAA+ ATPases that assemble into ring-shaped oligomers, often hexamers, with a central channel (Guo et al. Molecular Cell, 2:149). This chapter focuses on the most recent findings in the bacteriophage ϕ29 DNA packaging nanomotor to address this intriguing notion. Almost all dsDNA viruses are composed entirely of protein, but in the unique case of ϕ29, packaging RNA (pRNA) plays an intermediate role in the packaging process. Evidence revealed that DNA packaging is accomplished via a "push through one-way valve" mechanism. The ATPase gp16 pushes dsDNA through the connector channel section by section into the procapsid. The dodecameric connector channel functions as a one-way valve that only allows dsDNA to enter but not exit the procapsid during DNA packaging. Although the roles of the ATPase gp16 and the motor connector channel are separate and independent, pRNA bridges these two components to ensure the coordination of an integrated motor. ATP induces a conformational change in gp16, leading to its stronger binding to dsDNA. Furthermore, ATP hydrolysis led to the departure of dsDNA from the ATPase/dsDNA complex, an action used to push dsDNA through the connector channel. It was found unexpectedly that by mutating the basic lysine rings of the connector channel or by changing the pH did not measurably impair DNA translocation or affect the one-way traffic property of the channel, suggesting that the positive charges in the lysine ring are not essential in gearing the dsDNA. The motor channel exercises three discrete, reversible, and controllable steps of gating, with each step altering the channel size by 31% to control the direction of translocation of dsDNA. Many DNA packaging models have been contingent upon the number of base pairs packaged per ATP relative to helical turns for B-type DNA. Both 2 and 2.5 bp per ATP have been used to argue for four, five, or six discrete steps of DNA translocation. The "push through one-way valve" mechanism renews the perception of dsDNA packaging energy calculations and provides insight into the discrepancy between 2 and 2.5 bp per ATP. Application of the DNA packaging motor in nanotechnology and nanomedicine is also addressed. Comparison with nine other DNA packaging models revealed that the "push through one-way valve" is the most agreeable mechanism to interpret most of the findings that led to historical models. The application of viral DNA packaging motors is also discussed.
Nanobiotechnology involves the creation, characterization, and modification of organized nanomaterials to serve as building
blocks for constructing nanoscale devices in technology and medicine. Living systems contain a wide variety of nanomachines
and highly ordered structures of macromolecules. The novelty and ingenious design of the bacterial virus phi29 DNA packaging
motor and its parts inspired the synthesis of this motor and its components as biomimetics. This 30-nm nanomotor uses six
copies of an ATP-binding pRNA to gear the motor. The structural versatility of pRNA has been utilized to construct dimers,
trimers, hexamers, and patterned superstructures via the interaction of two interlocking loops. The approach, based on bottom-up
assembly, has also been applied to nanomachine fabrication, pathogen detection and the delivery of drugs, siRNA, ribozymes,
and genes to specific cells in vitro and in vivo. Another essential component of the motor is the connector, which contains 12 copies of a protein gp10 to form a 3.6-nm central
channel as a path for DNA. This article will review current studies of the structure and function of the phi29 DNA packaging
motor, as well as the mechanism of motion, the principle of in vitro construction, and its potential nanotechnological and medical applications.
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.
The structure of an induced macromolecular assembly was characterized and found to consist of an ordered heptameric arrangement of recombinant phi29 gp10 connector molecules. Insertion of an N-terminal Strep-II/His(6) tag to the connectors led to the spontaneous formation of large nanoparticles that were distinct from free, wild-type phi29 connectors in both size and symmetry elements. The determination of single-molecule tomograms and image-averaged reconstructions allowed for the stoichiometric and topological characterization of the ordered assemblage, revealing that the nanoparticle is composed of five equatorial connectors arranged with pseudo-5-fold rotational symmetry, capped on its ends by two polar connectors. Additionally, all seven connectors are oriented with their narrower N-terminal necks into the nanoparticle core and wider C-terminal ends out toward the nanoparticle surface, a geometric arrangement accommodated by the shape complementarity of the conical connector profiles. A significant amount of conformational heterogeneity was detected, ranging from changes in overall nanoparticle diameter, to tilting of individual connectors, to variations in connector stoichiometry. Nevertheless, a stable, heptameric nanoparticle was resolved, revealing the significant potential of guided, peptide-mediated supramolecular self-assembly. With this construct, we anticipate the further design of variable N-terminal tags to allow for the generation of nanoparticles with tailored connector stoichiometry and topological arrangements. By modifying the surface-exposed C-terminal ends with application-appropriate moieties, the consistent structure and compact nature of these nanoparticles may prove beneficial in nanotechnological and nanomedical approaches.
Biological systems contain highly-ordered macromolecular structures with diverse functions, inspiring their utilization in nanotechnology. A motor allows linear dsDNA viruses to package their genome into a preformed procapsid. The central component of the motor is the portal connector that acts as a pathway for the translocation of dsDNA. The elegant design of the connector and its channel motivates its application as an artificial nanopore (Nature Nanotechnology, 4, 765-772). Herein, we demonstrate the robust characteristics of the connector of the bacteriophage phi29 DNA packaging motor by single pore electrophysiological assays. The conductance of each pore is almost identical and is perfectly linear with respect to the applied voltage. Numerous transient current blockade events induced by dsDNA are consistent with the dimensions of the channel and dsDNA. Furthermore, the connector channel is stable under a wide range of experimental conditions including high salt and pH 2-12. The robust properties of the connector nanopore made it possible to develop a simple reproducible approach for connector quantification. The precise number of connectors in each sheet of the membrane was simply derived from the slopes of the plot of voltage against current. Such quantifications led to a reliable real time counting of DNA passing through the channel. The fingerprint of DNA translocation in this system has provided a new tool for future biophysical and physicochemical characterizations of DNA transportation, motion, and packaging.
Biological pores have been used to study the transport of DNA and other molecules, but most pores have channels that allow only the movement of small molecules and single-stranded DNA and RNA. The bacteriophage phi29 DNA-packaging motor, which allows double-stranded DNA to enter the virus during maturation and exit during an infection, contains a connector protein with a channel that is between 3.6 and 6 nm wide. Here we show that a modified version of this connector protein, when reconstituted into liposomes and inserted into planar lipid bilayers, allows the translocation of double-stranded DNA. The measured conductance of a single connector channel was 4.8 nS in 1 M KCl. This engineered and membrane-adapted phage connector is expected to have applications in microelectromechanical sensing, microreactors, gene delivery, drug loading and DNA sequencing.
‘Bottom-up fabrication’, which exploits the intrinsic properties of atoms and molecules to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by ‘top-down’ methods. The self-assembly of DNA molecules provides an attractive route towards this goal. Here I describe a simple method for folding long, single-stranded DNA molecules into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide ‘staple strands’ to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diameter and approximate desired shapes such as squares, disks and five-pointed stars with a spatial resolution of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton molecular complex).
Cell walls are an important structural component of prokaryotic organisms and essential for many aspects of their life. Particularly, the diverse structures of the outermost boundary layers strongly reflect adaptations of organisms to specific ecological and environmental conditions (6).
The self-organization of CdSe nanocrystallites into three-dimensional semiconductor quantum dot superlattices (colloidal crystals)
is demonstrated. The size and spacing of the dots within the superlattice are controlled with near atomic precision. This
control is a result of synthetic advances that provide CdSe nanocrystallites that are monodisperse within the limit of atomic
roughness. The methodology is not limited to semiconductor quantum dots but provides general procedures for the preparation
and characterization of ordered structures of nanocrystallites from a variety of materials.
We demonstrate the use of a one-dimensional template to control the shape of a two-dimensional array self-assembled from a minimal set of DNA tiles. A periodic single-stranded template seeds tile assembly. A unique vertex tile at the 5′ end of the template controls the positioning of edge and body tiles to create a wedge-shaped array. The vertex angle of the array is approximately 12°; edge lengths are of the order of 1 μm.Graphical abstract
As part of the viral infection cycle, viruses must package their newly
replicated genomes for delivery to other host cells. Bacteriophage
φ29 packages its 6.6-µm long, double-stranded DNA into a
42×54nm capsid by means of a portal complex that hydrolyses ATP.
This process is remarkable because entropic, electrostatic and bending
energies of the DNA must be overcome to package the DNA to
near-crystalline density. Here we use optical tweezers to pull on single
DNA molecules as they are packaged, thus demonstrating that the portal
complex is a force-generating motor. This motor can work against loads
of up to 57pN on average, making it one of the strongest molecular
motors reported to date. Movements of over 5µm are observed,
indicating high processivity. Pauses and slips also occur, particularly
at higher forces. We establish the force-velocity relationship of the
motor and find that the rate-limiting step of the motor's cycle is force
dependent even at low loads. Notably, the packaging rate decreases as
the prohead is filled, indicating that an internal force builds up to
~50pN owing to DNA confinement. Our data suggest that this force may be
available for initiating the ejection of the DNA from the capsid during
infection.
Many nucleic acid-binding proteins and the AAA+ family form hexameric rings, but the mechanism of hexamer assembly is unclear.
It is generally believed that the specificity in protein/RNA interaction relies on molecular contact through a surface charge
or 3D structure matching via conformational capture or induced fit. The pRNA of bacteriophage phi29 DNA-packaging motor also
forms a ring, but whether the pRNA ring is a hexamer or a pentamer is under debate. Here, single molecule studies elucidated
a mechanism suggesting the specificity and affinity in protein/RNA interaction relies on pRNA static ring formation. A combined
pRNA ring-forming group was very specific for motor binding, but the isolated individual members of the ring-forming group
bind to the motor nonspecifically. pRNA did not form a ring prior to motor binding. Only those RNAs that formed a static ring,
via the interlocking loops, stayed on the motor. Single interlocking loop interruption resulted in pRNA detachment. Extension
or reduction of the ring circumference failed in motor binding. This new mechanism was tested by redesigning two artificial
RNAs that formed hexamer and packaged DNA. The results confirmed the stoichiometry of pRNA on the motor was the common multiple
of two and three, thus, a hexamer.
DNA's remarkable molecular recognition properties and structural features make it one of the most promising templates to pattern materials with nanoscale precision. The emerging field of DNA nanotechnology strips this molecule from any preconceived biological role and exploits its simple code to generate addressable nanostructures in one, two, and three dimensions. These structures have been used to precisely position proteins, nanoparticles, transition metals, and other functional components into deliberately designed patterns. They can also act as templates for the growth of nanowires, aid in the structural determination of proteins, and provide new platforms for genomics applications. The field of DNA nanotechnology is growing in a number of directions, carrying with it the promise to substantially affect materials science and biology.
Molecular self-assembly is a promising approach to the preparation of nanostructures. DNA, in particular, shows great potential to be a superb molecular system. Synthetic DNA molecules have been programmed to assemble into a wide range of nanostructures. It is generally believed that rigidities of DNA nanomotifs (tiles) are essential for programmable self-assembly of well defined nanostructures. Recently, we have shown that adequate conformational flexibility could be exploited for assembling 3D objects, including tetrahedra, dodecahedra, and buckyballs, out of DNA three-point star motifs. In the current study, we have integrated tensegrity principle into this concept to assemble well defined, complex nanostructures in both 2D and 3D. A symmetric five-point-star motif (tile) has been designed to assemble into icosahedra or large nanocages depending on the concentration and flexibility of the DNA tiles. In both cases, the DNA tiles exhibit significant flexibilities and undergo substantial conformational changes, either symmetrically bending out of the plane or asymmetrically bending in the plane. In contrast to the complicated natures of the assembled structures, the approach presented here is simple and only requires three different component DNA strands. These results demonstrate that conformational flexibility could be explored to generate complex DNA nanostructures. The basic concept might be further extended to other biomacromolecular systems, such as RNA and proteins.
• icosahedron
• three-dimensional
• polyhedron
• cryo-EM
• molecular cages
The bacteriophage phi 29 DNA-gene product 3 complex (DNA-gp3) has been efficiently packaged into proheads in a completely defined in vitro system. The phi 29 DNA packaging protein gp16, the product of gene 16, was overproduced in Escherichia coli and purified to near homogeneity. The purified gp16 packaged 23% of the DNA-gp3 added to purified proheads in the defined mixture, while gp16 in an extract of phage-infected cells packaged 26% of the DNA-gp3. No host proteins were required in the defined system. ATP-dependent packaging of DNA-gp3 in the defined system was optimal with approximately equal to 100 copies of gp16 per DNA-gp3, an amount similar to the production of gp16 per DNA-gp3 in phi 29-infected cells.
The head-tail connector of bacteriophage phi29, an oligomer of gene product 10 (gp10), was crystallized into various forms. The most useful of these were an orthorhombic P22(1)2(1) form (unit-cell parameters a = 143.0, b = 157.0, c = 245.2 A), a monoclinic C2 form (a = 160.7, b = 143.6, c = 221.0 A, beta = 97.8 degrees ) and another monoclinic C2 form (a = 177.0, b = 169.1, c = 185.2 A, beta = 114.1 degrees ). Frozen crystals diffracted to about 3.2 A resolution. There is one connector per crystallographic asymmetric unit in each case. Rotation functions show the connector to be a dodecamer. Translation functions readily determined the position of the 12-fold axis in each unit cell. The structure is being determined by 12-fold electron-density averaging within each crystal and by averaging between the various crystal forms.
Recent work has demonstrated the self-assembly of designed periodic two-dimensional arrays composed of DNA tiles, in which the intermolecular contacts are directed by 'sticky' ends. In a mathematical context, aperiodic mosaics may be formed by the self-assembly of 'Wang' tiles, a process that emulates the operation of a Turing machine. Macroscopic self-assembly has been used to perform computations; there is also a logical equivalence between DNA sticky ends and Wang tile edges. This suggests that the self-assembly of DNA-based tiles could be used to perform DNA-based computation. Algorithmic aperiodic self-assembly requires greater fidelity than periodic self-assembly, because correct tiles must compete with partially correct tiles. Here we report a one-dimensional algorithmic self-assembly of DNA triple-crossover molecules that can be used to execute four steps of a logical (cumulative XOR) operation on a string of binary bits.
We report a technique for investigating nucleation and growth confined to nanometer scale surfaces. Lithographic and etching processes were used to create arrays of 100 and 150 nm holes through a thin SiO2 layer onto Si(100). Ge dots were nucleated and grown to a few nanometers in diameter within the patterned wells. Transmission electron and atomic force microscopic analyses revealed the presence of 0−1 Ge quantum dots in each of the 100 nm wells and 2−4 dots in the 150 nm wells. For the latter case, size−distance correlations indicated the effective radius of the diffusion field around a growing Ge particle was much larger than for growth on an infinite surface.
Colloidal crystals are three-dimensional periodic structures formed from
small particles suspended in solution. They have important technological
uses as optical filters1-3, switches4 and
materials with photonic band gaps5,6, and they also provide
convenient model systems for fundamental studies of crystallization and
melting7-10. Unfortunately, applications of colloidal
crystals are greatly restricted by practical difficulties encountered in
synthesizing large single crystals with adjustable crystal
orientation11. Here we show that the slow sedimentation of
colloidal particles onto a patterned substrate (or template) can direct
the crystallization of bulk colloidal crystals, and so permit tailoring
of the lattice structure, orientation and size of the resulting
crystals: we refer to this process as 'colloidal epitaxy'. We also show
that, by using silica spheres synthesized with a fluorescent
core12,13, the defect structures in the colloidal crystals
that result from an intentional lattice mismatch of the template can be
studied by confocal microscopy14. We suggest that colloidal
epitaxy will open new ways to design and fabricate materials based on
colloidal crystals and also allow quantitative studies of heterogeneous
crystallization in real space.
Large three-dimensional particle arrangements with lattice constants of the order of the wavelength of light, or colloidal crystals, are of interest to study, e.g., crystallization and defects, model porous media. In addition, they have fascinating optical properties. It has been shown previously, that it is possible to grow large single crystals with charged particles by fine-tuning the crystallization through the volume fraction. At very low volume fractions the nucleation rate can be kept small enough to allow large, albeit low density, crystals to form. Particles with hard-sphere interactions form much denser assemblies under the influence of a gravitational field. The hexagonally close-packed layers, which form perpendicular to the direction of gravity, are, however, stacked randomly and thus do not form a true crystal. Recently we have shown (A. van Blaaderen, R. Ruel, and P. Wiltzius, Nature, 385, 321 (1997); A. van Blaaderen and P. Wiltzius, Adv. Mat., 9, 833 (1997)) that we can grow large, well-oriented high density crystals with hard-spheres. We created FCC lattice planes of holes close to the micrometer particle size in a polymer layer with thickness close to the particle radius. Subsequently, we let the crystals grow on top of the template, by gravitational settling of monodisperse silica spheres, from a dilute dispersion. Once the first layer of spheres was crystallized under the influence of the template it can act as its own template. Pure FCC crystals were formed as large as the template and several thousand layers thick both in the (100) direction and in the (110) direction. These do not have the twinning problem that the dense (111) direction has leading to random stacking. Other methods of templating will be discussed.
Communication: Stable nanostructured networks of quantum-sized particles have been prepared (see Figure) using self-assembly techniques. Here, two new techniques are reported for the preparation of non-metallic composite materials comprising nanometer-sized gold particles self assembled into a 3-D network by means of organic dithiols.
The authors thank A. Wilkinson for discussion of early X-ray measurements; W. B. Carter for permission to mention photoelectron spectroscopic results, M. Duncan, J. Amster, T. P. Martin, and S. Frank for assistance in confirming and extending the mass spectrometry results; M. Shafigullin for help in analyzing structural models; L. Saldana for performing spectroscopic and stability measurements; R. Whyman for stimulating discussions; and R. Andres for communication of results prior to publication. Financial support has been provided by the Georgia Tech Research Foundation, the Packard Foundation and the Office of Naval Research (to RLW), and the U.S. Department of Energy, the National Science Foundation, and the Air Force Office of Scientific Research (to CC, WDL, and UL). Calculations were performed on CRAY computers at the National Energy Research Supercomputer Center, Livermore, California, and at the GIT Center for Computational Materials Science.
Three-dimensional quantum dot superlattices with fcc structure have been formed using reverse micelles and self-organization of Ag2S nanocrystals. It is reported that the size distribution of the nanoparticles can be narrowed by a chemical treatment of the interface and by particle extraction from the micelles. The formation of well-defined tow- or three-dimensional ordered arrays is required for the development of new types of optical gratings, optical filters, antireflective surface coatings, data storage systems, and microelectronics.
The technique for fabrication of thin textured carbon films is described. These textured films are made by a carbon/Victawet evaporation on a textured silicon substrate. This process lends itself to reproductions of dozens of textured films from the same master template.
Close-packed planar arrays of nanometer-diameter metal clusters that are covalently linked to each other by rigid, double-ended
organic molecules have been self-assembled. Gold nanocrystals, each encapsulated by a monolayer of alkyl thiol molecules,
were cast froma colloidal solution onto a flat substrate to form a close-packed cluster monolayer. Organic interconnects (aryl
dithiols or aryl di-isonitriles) displaced the alkyl thiol molecules and covalently linked adjacent clusters in the monolayer
to form a two-dimensional superlattice of metal quantum dots coupled by uniform tunnel junctions. Electrical conductance through
such a superlattice of 3.7-nanometer-diameter gold clusters, deposited on a SiO2 substrate in the gap between two gold contacts
and linked by an aryl di-isonitrile [1,4-di(4-isocyanophenylethynyl)-2-ethylbenzene], exhibited nonlinear Coulomb charging
behavior.
Self-assembled cylindrical particles of wild type and recombinant tobacco mosaic virus (TMV) were used as organic templates for the controlled deposition and organization of Pt, Au, or Ag nanoparticles. Chemical reduction of [PtCl6]2- or [AuCl4]- complexes at acidic pH gave rise to the specific decoration of the external surface of wild-type TMV rods with metallic nanoparticles less than 10 nm in size. In contrast, photochemical reduction of Ag(I) salts at pH 7 resulted in nucleation and constrained growth of discrete Ag nanoparticles aligned within the 4 nm-wide internal channel. The number of encapsulated nanoparticles increased when Ag benzoate rather than Ag nitrate was used due to reduced supersaturation associated with the lower Ag/benzoate redox couple, which enhanced the surface-templating effect of the channel wall carboxylates compared with nucleation in solution. Similar experiments using a mutant TMV with reduced negative charge along the central cavity confirmed that glutamic and aspartate acid groups were involved in site-specific deposition. Our results suggest that it should be possible to prepare 1-D arrays for a wide range of inorganic quantum dots by molecular engineering of the internal and external surfaces of self-assembled TMV tubules.
A method is described for attaching semiconductor nanocrystals to metal surfaces using self-assembled difunctional organic monolayers as bridge compounds. Three different techniques are presented. Two rely on the formation of self-assembled monolayers on gold and aluminum in which the exposed tail groups are thiols. When exposed to heptane solutions of cadmium-rich nanocrystals, these free thiols bind the cadmium and anchor it to the surface. The third technique attaches nanocrystals already coated with carboxylic acids to freshly cleaned aluminum. The nanocrystals, before deposition on the metals, were characterized by ultraviolet-visible spectroscopy, X-ray powder diffraction, resonance Raman scattering, transmission electron microscopy (TEM), and electron diffraction. Afterward, the nanocrystal films were characterized by resonance Raman scattering, Rutherford back scattering (RBS), contact angle measurements, and TEM. All techniques indicate the presence of quantum confined clusters on the metal surfaces with a coverage of approximately 0.5 monolayers. These samples represent the first step toward synthesis of an organized assembly of clusters as well as allow the first application of electron spectroscopies to be completed on this type of cluster. As an example of this, the first X-ray photoelectron spectra of semiconductor nanocrystals are presented. 51 refs., 17 figs.
The Langmuir-Blodgett (LB) technique is used to deposit monolayers of nearly monodisperse nanometer-size CdSe crystallites (quantum dots) onto various substrates. Size-selected CdSe nanocrystallites capped with trioctylphosphine oxide are directly applied onto the water surface of a LB trough and serve as the LB-active species. Absorption and luminescence studies of monolayers transferred onto glass slides indicate that the monolayers retain the general optical properties of isolated crystallites. Transmission electron micrographs of monolayers on amorphous carbon show the formation of two-dimensional hexagonal close-packed domains.
Deoxyribonucleic acid (DNA)-derivatized gold nanowires were discussed as the building blocks of surface assemblies. Oligonucleotides were adsorbed as monolayer coatings on the wires through gold-thiol linkages. Absorption and fluorescence spectroscopy were employed to quantify the amount of DNA bound. The nanowires were modified with single stranded (ss) DNA at specific sites with the remaining wire covered by passivating monolayers. The modification resulted in easy implementation of the site-specific DNA directed assembly.
Materials scientists increasingly draw inspiration from the study of how biological systems fabricate materials under mild synthetic conditions by using self-assembled macromolecular templates. Containerlike protein architectures such as viral capsids and ferritin are examples of such biological templates. These protein cages have three distinct interfaces that can be synthetically exploited: the interior, the exterior, and the interface between subunits. The subunits that comprise the building blocks of these structures can be modified both chemically and genetically in order to impart designed functionality to different surfaces of the cage. Therefore, the cages possess a great deal of synthetic flexibility, which allows for the introduction of multifunctionality in a single cage. In addition, hierarchical assembly of the functionalized cages paves the way for development of a new class of materials with a wide range of applications from electronics to biomedicine.
A computer program system, CRISP, for crystallographic image processing (CIP), has been developed. CRISP runs on a standard personal computer (PC), and is considerably faster than previous systems for CIP. High-resolution electron microscopy (HREM) images are digitized by a CCD camera and directly transferred to the PC via a frame grabber. CRISP has been designed with strong emphasis on user friendliness. Thus, installation takes only a few minutes, a full processing of an HREM image takes less than 20 minutes, all operations are carried out with a mouse, and all the crystallography needed for 2D analysis of images is programmed into CRISP.
The mechanism of DNA packaging by dsDNA viruses is not well understood in any system. In bacteriophage P22 only five genes are required for successful condensation of DNA within the capsid. The products of three of these genes, the portal, scaffolding, and coat proteins, are structural components of the precursor particle, and two, the products of genes 2 and 3, are not. The scaffolding protein is lost from the structure during packaging, and only the portal and coat proteins are present in the mature virus particle. These five genes map in a contiguous cluster at the left end of the P22 genetic map. Three additional genes, 4, 10, and 26, are required for stabilization of the condensed DNA within the capsid. In this report we present the nucleotide sequence of 7461 bp of P22 DNA that contains the five genes required for DNA condensation, as well as a nonessential open reading frame (ORF109), gene 4, and a portion of gene 10. N-terminal amino acid sequencing of the encoded proteins accurately located the translation starts of six genes in the sequence. Despite the fact that most of these proteins have striking analogs in the other dsDNA bacteriophage groups, which perform highly analogous functions, no amino acid sequence similarity between these analogous proteins has been found, indicating either that they diverged a very long time ago or that they are the products of spectacular convergent evolution.
The connector protein of bacteriophage T3, p8, has been overexpressed in Escherichia coli. Purification of the oligomers built by several copies of p8 reveals a mixed population of dodecamers and tridecamers. The percentages of these two types of oligomers differ in every culture growth, indicating that assembly of this protein depends upon the conditions of the expression system. Those cultures that generated a majority of dodecamers allowed, after purification of the connectors, the two-dimensional crystallization of the dodecamers in a tetragonal arrangement, while the tridecamers did not form crystals. The processing and averaging of several images of frozen-hydrated crystals and their internal phase comparison shows that the crystals are arranged in a P4212 space group, with cell unit dimensions of 165 × 165 Å. The three-dimensional reconstruction generated with images of crystals ranging from 0° to 60° tilt reveals a wide domain surrounded by 12 protrusions and a narrow domain that serves to interact with the tail of the bacteriophage. A channel runs along the connector wide enough to allow the translocation of a double-stranded DNA molecule into the prohead. The general structure of the T3 connector is very similar to those obtained for other nonrelated bacteriophages and strongly suggests that the shape of this important viral structure is intimately related to its function.
Synthetic biology is a rapidly growing field that has emerged in a global, multidisciplinary effort among biologists, chemists, engineers, physicists, and mathematicians. Broadly, the field has two complementary goals: To improve understanding of biological systems through mimicry and to produce bio-orthogonal systems with new functions. Here we review the area specifically with reference to the concept of synthetic biology space, that is, a hierarchy of components for, and approaches to generating new synthetic and functional systems to test, advance, and apply our understanding of biological systems. In keeping with this issue of Current Opinion in Structural Biology, we focus largely on the design and engineering of biomolecule-based components and systems.
DNA has many physical and chemical properties that make it a powerful material for molecular constructions at the nanometer length scale. In particular, its ability to form duplexes and other secondary structures through predictable nucleotide-sequence-directed hybridization allows for the design of programmable structural motifs which can self-assemble to form large supramolecular arrays, scaffolds, and even mechanical and logical nanodevices. Despite the large variety of structural motifs used as building blocks in the programmed assembly of supramolecular DNA nanoarchitectures, the various modules share underlying principles in terms of the design of their hierarchical configuration and the implemented nucleotide sequences. This Review is intended to provide an overview of this fascinating and rapidly growing field of research from the structural design point of view.
Nanomaterials, such as metal or semiconductor nanoparticles and nanorods, exhibit similar dimensions to those of biomolecules, such as proteins (enzymes, antigens, antibodies) or DNA. The integration of nanoparticles, which exhibit unique electronic, photonic, and catalytic properties, with biomaterials, which display unique recognition, catalytic, and inhibition properties, yields novel hybrid nanobiomaterials of synergetic properties and functions. This review describes recent advances in the synthesis of biomolecule-nanoparticle/nanorod hybrid systems and the application of such assemblies in the generation of 2D and 3D ordered structures in solutions and on surfaces. Particular emphasis is directed to the use of biomolecule-nanoparticle (metallic or semiconductive) assemblies for bioanalytical applications and for the fabrication of bioelectronic devices.
DNA and protein have been extensively scrutinized for feasibility as parts in nanotechnology, but another natural building block, RNA, has been largely ignored. RNA can be manipulated to form versatile shapes, thus providing an element of adaptability to DNA nanotechnology, which is predominantly based upon a double-helical structure. The DNA-packaging motor of bacterial virus phi29 contains six DNA-packaging RNAs (pRNA), which together form a hexameric ring via loop/loop interaction. Here we report that this pRNA can be redesigned to form a variety of structures and shapes, including twins, tetramers, rods, triangles, and 3D arrays several microns in size via interaction of programmed helical regions and loops. Three dimensional RNA array formation required a defined nucleotide number for twisting of the interactive helix and a palindromic sequence. Such arrays are unusually stable and resistant to a wide range of temperatures, salt concentrations, and pH.
通讯作者地址: Feng, PY (通讯作者), Univ Calif Riverside, Dept Chem, Riverside, CA 92521 USA 地址: 1. Univ Calif Riverside, Dept Chem, Riverside, CA 92521 USA 2. Univ Calif Santa Barbara, Dept Chem, Santa Barbara, CA 93106 USA
Recent work has demonstrated the self-assembly of designed periodic
two-dimensional arrays composed of DNA tiles, in which the
intermolecular contacts are directed by `sticky' ends. In a mathematical
context, aperiodic mosaics may be formed by the self-assembly of `Wang'
tiles, a process that emulates the operation of a Turing machine.
Macroscopic self-assembly has been used to perform computations; there
is also a logical equivalence between DNA sticky ends and Wang tile
edges. This suggests that the self-assembly of DNA-based tiles could be
used to perform DNA-based computation. Algorithmic aperiodic
self-assembly requires greater fidelity than periodic self-assembly,
because correct tiles must compete with partially correct tiles. Here we
report a one-dimensional algorithmic self-assembly of DNA
triple-crossover molecules that can be used to execute four steps of a
logical (cumulative XOR) operation on a string of binary bits.
The ATPase activity of the DNA packaging protein gp16 (gene product 16) of bacteriophage phi 29 was studied in the completely defined in-vitro assembly system. ATP was hydrolyzed to ADP and Pi in the packaging reaction that included purified proheads, DNA-gp3 and gp16. Approximately one molecule of ATP was used in the packaging of 2 base-pairs of phi 29 DNA, or 9 X 10(3) ATP molecules per virion. The hydrolysis of ATP by gp16 was both prohead and DNA-gp3 dependent. gp16 contained both the "A-type" and the "B-type" ATP-binding consensus sequences (Walker et al., 1982) and the predicted secondary structure for ATP binding. The A-type sequence of gp16 was "basic-hydrophobic region-G-X2-G-X-G-K-S-X7-hydrophobic", and similar sequences were found in the phage DNA packaging proteins gpA of lambda, gp19 of T7 and gp17 of T4. Having both the ATP-binding and potential magnesium-binding domains, all of these proteins probably function as ATPases and may have common prohead-binding capabilities. The phi 29 protein gp3, covalently bound to the DNA, may be analogous in function to proteins gpNul of lambda and gpl of phi 21 that bind the DNA.
A small RNA of Bacillus subtilis bacteriophage phi 29 is shown to have a novel and essential role in viral DNA packaging in vitro. This requirement for RNA in the encapsidation of viral DNA provides a new dimension of complexity to the attendant protein-DNA interactions. The RNA is a constituent of the viral precursor shell of the DNA-packaging machine but is not a component of the mature virion. Studies of the sequential interactions involving this RNA molecule are likely to provide new insight into the structural and possible catalytic roles of small RNA molecules. The phi 29 assembly in extracts and phi 29 DNA packaging in the defined in vitro system were strongly inhibited by treatment with the ribonucleases A or T1. However, phage assembly occurred normally in the presence of ribonuclease A that had been treated with a ribonuclease inhibitor. An RNA of approximately 120 nucleotides co-purified with the phi 29 precursor protein shell (prohead), and this particle was the target of ribonuclease action. Removal of RNA from the prohead by ribonuclease rendered it inactive for DNA packaging. By RNA-DNA hybridization analysis, the RNA was shown to originate from a viral DNA segment very near the left end of the genome, the end packaged first during in vitro assembly.
The three-dimensional reconstruction of the connector of bacteriophage phi 29 has been obtained from tilt series of negatively stained tetragonal ordered aggregates under low-dose conditions and up to a resolution of (1/1.8) nm-1. These connectors are built up as dodecamers of only one structural polypeptide (p10). Two connectors form the crystal unit cell, each one facing in the opposite direction with respect to the plane of the crystal and partially overlapping. The main features of the two connectors that build the unit cell were essentially the same, although they were negatively stained in slightly different ways, probably due to their situations with respect to the carbon-coated support grid. The main features of the phi 29 connector structure revealed by this three-dimensional reconstruction are: the existence of two clearly defined domains, one with a diameter of around 14 nm and the other narrower (diameter approximately equal to 7.5 nm); an inner hole running all along the structure (around 7 to 8 nm in height) with a cylindrical profile and an average diameter of 4 nm; a general 6-fold symmetry along the whole structure and a 12-fold one in the wider domain; a clockwise twist of the more contrasted regions of both domains from the narrower towards the wider domain (the direction of DNA encapsidation). These features are compatible with an active role for the connector in the process of DNA packaging.
A computer graphic display method that produces two-dimensional perspective views of three-dimensional objects is presented. The method is applied to the reconstruction at a resolution of 2.2 nanometers of the neck of bacteriophage phi 29, obtained from transmission electron micrographs processed by the direct Fourier method. The combined use of directed illumination, reflectance models, color, and different levels of transparency provides a powerful tool for a better interpretation of the three-dimensional structure, allowing improved correlation with genetic, structural, and biochemical data.
The phi 29 DNA restriction fragment HindIII-D, shown to contain gene 10 coding for the connector protein, has been cloned in plasmid pPLc28 under the control of the pL promoter of phage lambda. After heat induction to inactivate the lambda repressor, a protein with the electrophoretic mobility of the connector protein p10 was synthesized, accounting for about 30% of the total Escherichia coli protein after 3 h of induction. The 2205 nucleotide-long sequence of the cloned HindIII-D fragment has been determined. The sequenced region has an ORF coding for a protein of Mr 35881 that was shown to correspond to the connector protein by determination of the amino-terminal sequence of purified protein p10. Features of the nucleotide sequence and the amino acid sequence of protein p10 are discussed.
A purification method is described for gene 20 product, which is a quantitatively minor, but essential, protein of the bacteriophage T4 head. During purification and in response to different solvent conditions the protein behaves as if it were hydrophobic. Its structure has been studied by conventional and scanning transmission electron microscopy and sedimentation analysis. Gene 20 product exists as a dodecameric structure with 12-fold rotational symmetry and a molecular weight of about 740,000 as measured by two independent methods (electron scattering and sedimentation equilibrium). It is composed of a hollow cylinder, 14 nm long and 7 nm diameter, and 12 radial protrusions, each measuring about 7 nm × 5 nm. Double rings and microcrystals of rings have been observed. The microcrystals measure about 1 μm and have a body-centered tetragonal type of lattice. The structure of the gene 20 product oligomer, in particular its symmetry, has consequences for the assembly of the T4 head, whose shell has 5-fold symmetry.
Patterning matter on the nanometre scale is an important objective of current materials chemistry and physics. It is driven by both the need to further miniaturize electronic components and the fact that at the nanometre scale, materials properties are strongly size-dependent and thus can be tuned sensitively. In nanoscale crystals, quantum size effects and the large number of surface atoms influence the, chemical, electronic, magnetic and optical behaviour. 'Top-down' (for example, lithographic) methods for nanoscale manipulation reach only to the upper end of the nanometre regime; but whereas 'bottom-up' wet chemical techniques allow for the preparation of mono-disperse, defect-free crystallites just 1-10 nm in size, ways to control the structure of nanocrystal assemblies are scarce. Here we describe a strategy for the synthesis of 'nanocrystal molecules', in which discrete numbers of gold nanocrystals are organized into spatially defined structures based on Watson-Crick base-pairing interactions. We attach single-stranded DNA oligonucleotides of defined length and sequence to individual nanocrystals, and these assemble into dimers and trimers on addition of a complementary single-stranded DNA template. We anticipate that this approach should allow the construction of more complex two- and three-dimensional assemblies.
Colloidal particles of metals and semiconductors have potentially useful optical, optoelectronic and material properties that derive from their small (nanoscopic) size. These properties might lead to applications including chemical sensors, spectroscopic enhancers, quantum dot and nanostructure fabrication, and microimaging methods. A great deal of control can now be exercised over the chemical composition, size and polydispersity of colloidal particles, and many methods have been developed for assembling them into useful aggregates and materials. Here we describe a method for assembling colloidal gold nanoparticles rationally and reversibly into macroscopic aggregates. The method involves attaching to the surfaces of two batches of 13-nm gold particles non-complementary DNA oligonucleotides capped with thiol groups, which bind to gold. When we add to the solution an oligonucleotide duplex with 'sticky ends' that are complementary to the two grafted sequences, the nanoparticles self-assemble into aggregates. This assembly process can be reversed by thermal denaturation. This strategy should now make it possible to tailor the optical, electronic and structural properties of the colloidal aggregates by using the specificity of DNA interactions to direct the interactions between particles of different size and composition.
Crystalline cell surface layers (S-layers) composed of planar assemblies of protein or glycoprotein subunits are one of the most commonly observed cell envelope structures of bacteria and archaea. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at liquid-surface interfaces, including lipid films, on liposomes and on solid supports. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattices are aligned in well-defined positions and orientations. These characteristic features of S-layers have led to various applications in biotechnology, vaccine development, diagnostics, biomimetics and molecular nanotechnology.
The internal symmetry of the connector or portal particle from the double-stranded DNA bacteriophage phi29 has been examined by X-ray crystallography. This large multimeric structure (420 kDa) is built up by a number of identical subunits of the p10 protein. It connects the head of the virus with the tail and plays a central role in the prohead assembly and DNA packaging. For the first time a bacteriophage connector has been crystallized and X-ray data have been collected up to a resolution of 3.2 A. A self-rotation function has been calculated, unambigously revealing the 12-fold symmetry of the particle and its orientation in the crystal lattice. The orientation has been confirmed by calculating a cross-rotation function using a low resolution model based on electron microscopy reconstructions.
Head-tail connectors are viral substructures that are very important in the viral morphogenetic cycle, having roles in the formation of the precursor capsid (prohead), DNA packaging, tail binding to the mature head and in the infection process. Structural information on the connector would, therefore, help us to understand how this structure is related to a multiplicity of functions.
Recombinant bacteriophage phi29 connectors have been crystallized in two-dimensional aggregates. An average projection image and a three-dimensional map have been obtained at 8 A and 10 A resolution, respectively, from untilted and tilted images of vitrified specimens of the two-dimensional crystals. The average projection image reveals a central mass surrounding a channel with 12 appendages protruding from the central mass. The three-dimensional map reveals a wide domain surrounded by 12 appendages that interact with the prohead vertex, and a narrow domain that interacts with the bacteriophage tail. At the junction of the two domains, 12 smaller appendages are visualized. A channel runs along the axis of the connector structure and is sufficiently wide to allow a double-stranded DNA molecule to pass through.
The propeller-like structure of the phi29 connector strengthens the notion of the connector rotating during DNA packaging. The groove formed by the two lanes of large and small appendages may act as a rail to prevent the liberation of the connector from the prohead vertex during rotation.
We present EMAN (Electron Micrograph ANalysis), a software package for performing semiautomated single-particle reconstructions from transmission electron micrographs. The goal of this project is to provide software capable of performing single-particle reconstructions beyond 10 A as such high-resolution data become available. A complete single-particle reconstruction algorithm is implemented. Options are available to generate an initial model for particles with no symmetry, a single axis of rotational symmetry, or icosahedral symmetry. Model refinement is an iterative process, which utilizes classification by model-based projection matching. CTF (contrast transfer function) parameters are determined using a new paradigm in which data from multiple micrographs are fit simultaneously. Amplitude and phase CTF correction is then performed automatically as part of the refinement loop. A graphical user interface is provided, so even those with little image processing experience will be able to begin performing reconstructions. Advanced users can directly use the lower level shell commands and even expand the package utilizing EMAN's extensive image-processing library. The package was written from scratch in C++ and is provided free of charge on our Web site. We present an overview of the package as well as several conformance tests with simulated data.