'Scanning tunnelling microscopy (STM) images of the quantum states of an artificial atomic defect structure in silicon. This structure was fabricated by using the STM to individually remove five hydrogen atoms from a hydrogen-terminated silicon (001) surface. The absence of the hydrogen atoms creates “dangling bond” states that interact to form extended, artificial molecular orbitals. Only the imaging bias voltage has been changed in the three images shown (from left to right, -1.4, +1.4, and +1.8 Volts).' (credit: London Centre for Nanotechnology)

We have previously speculated here whether the continued improvement of technology to place single atoms on silicon with atomic precision for the purpose of developing practical quantum computers would also lead to more general methods of atomically precise or molecular manufacturing. That speculation remains open, but we note that the field of atomically precise quantum engineering continues to advance. A hat tip to ScienceDaily for reprinting this University College London news release “Building quantum states with individual silicon atoms“:

By introducing individual silicon atom ‘defects’ using a scanning tunnelling microscope, scientists at the London Centre for Nanotechnology have coupled single atoms to form quantum states.

Published today in Nature Communications [open access paper: Quantum engineering at the silicon surface using dangling bonds], the study demonstrates the viability of engineering atomic-scale quantum states on the surface of silicon – an important step toward the fabrication of devices at the single-atom limit.

Advances in atomic physics now allow single ions to be brought together to form quantum coherent states. However, to build coupled atomic systems in large numbers, as required for applications such as quantum computing, it is highly desirable to develop the ability to construct coupled atomic systems in the solid state.

Semiconductors, such as silicon, routinely display atomic defects that have clear analogies with trapped ions. However, introducing such defects deterministically to observe the coupling between extended systems of individual defects has so far remained elusive.

Now, LCN scientists have shown that quantum states can be engineered on silicon by creating interacting single-atom defects. Each individual defect consisted of a silicon atom with a broken, or “dangling”, bond. During this study, these single-atom defects were created in pairs and extended chains, with each defect separated by just under one nanometer.

Importantly, when coupled together, these individual atomic defects produce extended quantum states resembling artificial molecular orbitals. Just as for a molecule, each structure exhibited multiple quantum states with distinct energy levels.

The visibility of these states to the scanning tunneling microscope could be tuned through the variation of two independent parameters – the voltage applied to the imaging probe and its height above the surface.

The study was led by Dr Steven Schofield, who said: “We have created precise arrays of atomic defects on a silicon surface and demonstrated that they couple to form unique and interesting quantum states.”

He added: “The next step is to replicate these results in other material systems, for example using substitutional phosphorus atoms in silicon, which holds particular interest for quantum computer fabrication.”

Ongoing research at the LCN is exploring even more complex arrangements of these defects, including the incorporation of impurity atoms within the defect structures, which is expected to alter the symmetry of the defects (similar to the role of the nitrogen atom in the nitrogen-vacancy center defect in diamond).

Will this demonstrated ability of ‘quantum engineering’ dangling silicon atom bonds lead to applications to more general atomically precise manufacturing or productive nanosystems?
—James Lewis, PhD

Credit: Zhengyi Zhao, University of Kentucky

One of nature’s many types of molecular motors combines protein and RNA subunits to force viral (in this case, bacteriophage) DNA into a protein capsid. The understanding of the molecular mechanism by which this motor works has been advanced by the discovery that it revolves the DNA around a central channel, as the Earth revolves around the sun, rather than by rotating, as the Earth does about it axis. A hat tip to ScienceDaily for pointing to this research. From a University of Kentucky news release “Guo lab discovers new class of revolution biomotor and solves mystery in viral DNA packaging:”

Scientists at the University of Kentucky have cracked a 35-year-old mystery about the workings of natural “biomotors.” These molecular machines are serving as models for development of synthetic nanomotors that will someday pump therapeutic DNA, RNA or drugs into individual diseased cells.

The report, revealing the innermost mechanisms of these motors in a bacteria-killing virus and a “new way to move DNA through cells,” is being published online today in the journal ACS Nano.

The article, “Mechanism of One-Way Traffic of Hexameric Phi29 DNA Packaging Motor with Four Electropositive Relaying Layers Facilitating Anti-Parallel Revolution,” can be downloaded with free, open access from http://pubs.acs.org/doi/abs/10.1021/nn4002775.

Peixuan Guo, director of the UK Nanobiotechnology Center, and his colleagues explain that two motors have been found in nature: A linear motor and a rotating motor. Now they report discovery of a third type, a revolving molecular motor.

Guo points out that nanomotors will open the door to practical machines and other nanotechnology devices so small that 100,000 would fit across the width of a human hair. One major natural prototype for those development efforts has been the motor that packages DNA into the shell of bacteriophage phi29, a virus that infects and kills bacteria.

Guo’s own research team wants to embed a synthetic version of that motor into nanomedical devices that are injected into the body, travel to diseased cells and pump in medication. A major barrier in doing so has been uncertainty and controversy about exactly how the phi29 motor moves. Scientists thought that it worked by rotating or spinning in the same motion as the Earth turning once every 24 hours upon its own axis.

In their ACS Nano paper, Guo — with his team, Zhengyi Zhao, Emil Khisamutdinov, and Chad Schwartz — challenge that idea. Indeed, they discovered that the phi29 motor moves DNA without any rotational motion. The motor moves DNA with a revolving in the same motion as the Earth revolving around the sun in one orbit ever 365 days. The “revolution without rotation” model could resolve a big conundrum troubling the past 35 years of painstaking investigation of the mechanism of these viral DNA packaging motors, the report states.

Near-term application of artificial molecular motors based on this work are not difficult to imagine, such as in drug delivery or gene delivery for nanomedicine. Could motors like these be useful for more complicated molecular machine systems, such as running pulleys using DNA cables to transport components in primitive molecular manufacturing systems?
—James Lewis, PhD

Credit: Biodesign Institute

Of all of the paths toward molecular manufacturing, structural DNA nanotechnology seems to provide the most frequent and photogenic advances. By re-engineering the Holliday junction, the basic cross-over structure adapted to build complex structures from DNA, Prof. Hao Yan and his colleagues has been able to construct a variety of new wire frame and mesh structures. A hat tip to ScienceDaily for reprinting this Arizona State University news release “ASU scientists develop innovative twists to DNA nanotechnology“:

In a new discovery that represents a major step in solving a critical design challenge, Arizona State University Professor Hao Yan has led a research team to produce a wide variety of 2-D and 3-D structures that push the boundaries of the burgeoning field of DNA nanotechnology.

The field of DNA nanotechnology utilizes nature’s design rules and the chemical properties of DNA to self-assemble into an increasingly complex menagerie of molecules for biomedical and electronic applications. Some of the Yan lab’s accomplishments include building Trojan horse-like structures to improve drug delivery to cancerous cells, electrically conductive gold nanowires, single molecule sensors and programmable molecular robots.

With their bio-inspired architectural works, the group continues to explore the geometrical and physical limits of building at the molecular level.

“People in this field are very interested in making wire frame or mesh structures,” said Yan. “We needed to come up with new design principles that allow us to build with more complexity in three dimensions.”

In their latest twist to the technology, Yan’s team made new 2-D and 3-D objects that look like wire-frame art of spheres as well as molecular tweezers, scissors, a screw, hand fan, and even a spider web.

The Yan lab, which includes ASU Biodesign Institute colleagues Dongran Han, Suchetan Pal, Shuoxing Jiang, Jeanette Nangreave and assistant professor Yan Liu, published their results in the March 22 issue of Science [abstract].

The twist in their ‘bottom up,’ molecular Lego design strategy focuses on a DNA structure called a Holliday junction.

In nature, this cross-shaped, double-stacked DNA structure is like the 4-way traffic stop of genetics – where 2 separate DNA helices temporality meet to exchange genetic information. The Holliday junction is the crossroads responsible for the diversity of life on Earth, and ensures that children are given a unique shuffling of traits from a mother and father’s DNA.

In nature, the Holliday junction twists the double-stacked strands of DNA at an angle of about 60-degrees, which is perfect for swapping genes but sometimes frustrating for DNA nanotechnology scientists, because it limits the design rules of their structures.

“In principal, you can use the scaffold to connect multiple layers horizontally,” [which many research teams have utilized since the development of DNA origami by Cal Tech's Paul Rothemund in 2006]. However, when you go in the vertical direction, the polarity of DNA prevents you from making multiple layers,” said Yan. “What we needed to do is rotate the angle and force it to connect.”

Making the new structures that Yan envisioned required re-engineering the Holliday junction by flipping and rotating around the junction point about half a clock face, or 150 degrees. Such a feat has not been considered in existing designs.

“The initial idea was the hardest part,” said Yan. “Your mind doesn’t always see the possibilities so you forget about it. We had to break the conceptual barrier that this could happen.”

In the new study, by varying the length of the DNA between each Holliday junction, they could force the geometry at the Holliday junctions into an unconventional rearrangement, making the junctions more flexible to build for the first time in the vertical dimension. Yan calls the backyard barbeque grill-shaped structure a DNA Gridiron.

“We were amazed that it worked!” said Yan. “Once we saw that it actually worked, it was relatively easy to implement new designs. Now it seems easy in hindsight. If your mindset is limited by the conventional rules, it’s really hard to take the next step. Once you take that step, it becomes so obvious.”

The DNA Gridiron designs are programmed into a viral DNA, where a spaghetti-shaped single strand of DNA is spit out and folded together with the help of small ‘staple’ strands of DNA that help mold the final DNA structure. In a test tube, the mixture is heated, then rapidly cooled, and everything self-assembles and molds into the final shape once cooled. Next, using sophisticated AFM and TEM imaging technology, they are able to examine the shapes and sizes of the final products and determine that they had formed correctly.

This approach has allowed them to build multilayered, 3-D structures and curved objects for new applications.

“Most of our research team is now devoted toward finding new applications for this basic toolkit we are making,” said Yan. “There is still a long way to go and a lot of new ideas to explore. We just need to keep talking to biologists, physicists and engineers to understand and meet their needs.”

The video (computer simulation) of the sphere made from DNA, included in the news release, represents an impressive new capability. One thing I like about structural DNA nanotechnology is that every time I think they have just about exhausted the bag of tricks that DNA provides, someone proves me wrong. I look forward to seeing what else they come up with.
—James Lewis, PhD

Graphic representation of a 3-D atomic resolution screw dislocation in a platinum nanoparticle. Credit: Chien-Chun Chen and I-Sheng Chou, UCLA

Researchers from UCLA’s California NanoSystems Institute and Northwestern University have combined multiple imaging techniques to produce high quality 3D images of platinum nanoparticles, allowing advanced visualization of atomic-scale structural defects (an important advancement over X-ray crystallography).

The original 2012 work, published in Nature and posted by Jim Lewis here, used electron tomography to study 10-nm gold particles and was described at Phys.org:

…
“This is the first experiment where we can directly see local structures in three dimensions at atomic-scale resolution — that’s never been done before,” said Jianwei (John) Miao, a professor of physics and astronomy and a researcher with the California NanoSystems Institute (CNSI) at UCLA.
…
X-ray crystallography is a powerful technique for revealing the structure of perfect crystals, which are materials with an unbroken honeycomb of perfectly spaced atoms lined up as neatly as books on a shelf. Yet most structures existing in nature are non-crystalline, with structures far less ordered than their crystalline counterparts — picture a rock concert mosh pit rather than soldiers on parade.
…
Miao and his colleagues used a scanning transmission electron microscope to sweep a narrow beam of high-energy electrons over a tiny gold particle only 10 nanometers in diameter (almost 1,000 times smaller than a red blood cell). The nanoparticle contained tens of thousands of individual gold atoms, each about a million times smaller than the width of a human hair. These atoms interact with the electrons passing through the sample, casting shadows that hold information about the nanoparticle’s interior structure onto a detector below the microscope.

Miao’s team discovered that by taking measurements at 69 different angles, they could combine the data gleaned from each individual shadow into a 3-D reconstruction of the interior of the nanoparticle. Using this method, which is known as electron tomography, Miao’s team was able to directly see individual atoms and how they were positioned inside the specific gold nanoparticle.
…

The new study, using multiple imaging techniques, will be published in an upcoming issue of Nature, and includes a video showing three-dimensional volume renderings (available for viewing at Phys.org:

The authors describe being able to see how the atoms of a platinum nanoparticle—only 10 namometers in diameter—are arranged in three dimensions. They also identify how the atoms are arranged around defects in the platinum nanoparticle.
…
This novel method is a combination of three techniques: scanning transmission electron microscopy, equally sloped tomography (EST) and three-dimensional Fourier filtering. Compared to conventional CT, the combined method produces much higher quality 3-D images and allows the direct visualization of atoms inside the platinum nanoparticle in three dimensions.
…
“This is the first instance where the three-dimensional structure of dislocations in nanoparticles has been directly revealed at atomic resolution,” Ajayan said. “The elegant work demonstrates the power of electron tomography and leads to possibilities of directly correlating the structure of nanoparticles to properties, all in full 3-D view.” Defects can influence many properties of materials, and a technique for visualizing these structures at atomic resolution could lead to new insights beneficial to researchers in a wide range of fields.

-Posted by Stephanie C

(credit: Mary Leonard, University of Pennsylvania Biomedical Art & Design)

Research into using nanotechnology for improved drug delivery continues to advance as current nanoparticle technology is combined with increasingly more sophisticated biotechnology. One major problem with using nanoparticles for targeted drug delivery is that the patient’s immune system often clears the particle before they can be effective. A new approach uses a peptide derived from an important immune system molecule to fool the immune system. A commentary accompanying the publication (abstract) of the research in Science describes how short peptides from a human protein called CD47, which tells important immune system cells that cells or particles bearing the protein are human, not foreign, were used as a “passport” to get nanoparticles past the immune system. Additional details are supplied in a University of Pennsylvania news release “Penn Researchers Develop Protein ‘Passport’ That Helps Nanoparticles Get Past Immune System“:

… The research was conducted by professor Dennis Discher, graduate students Pia Rodriguez, Takamasa Harada, David Christian and Richard K. Tsai and postdoctoral fellow Diego Pantano … “From your body’s perspective,” Rodriguez said, “an arrowhead a thousand years ago and a pacemaker today are treated the same — as a foreign invader.

“We’d really like things like pacemakers, sutures and drug-delivery vehicles to not cause an inflammatory response from the innate immune system.”

The innate immune system attacks foreign bodies in a general way. Unlike the learned response of the adaptive immune system, which includes the targeted antibodies that are formed after a vaccination, the innate immune system tries to destroy everything it doesn’t recognize as being part of the body.

This response has many cellular components, including macrophages — literally “big eaters” — that find, engulf and destroy invaders. Proteins in blood serum work in tandem with macrophages; they adhere to objects in the blood stream and draw macrophages’ attention. If the macrophage determines these proteins are stuck to a foreign invader, they will eat it or signal other macrophages to form a barrier around it.

Drug-delivery nanoparticles naturally trigger this response, so researchers’ earlier attempts to circumvent it involved coating the particles with polymer “brushes.” These brushes stick out from the nanoparticle and attempt to physically block various blood serum proteins from sticking to its surface.

However, these brushes only slow down the macrophage-signaling proteins, so Discher and colleagues tried a different approach: Convincing the macrophages that the nanoparticles were part of the body and shouldn’t be cleared.

In 2008, Discher’s group showed that the human protein CD47, found on almost all mammalian cell membranes, binds to a macrophage receptor known as SIRPa in humans. Like a patrolling border guard inspecting a passport, if a macrophage’s SIRPa binds to a cell’s CD47, it tells the macrophage that the cell isn’t an invader and should be allowed to proceed on.

“There may be other molecules that help quell the macrophage response,” Discher said. “But human CD47 is clearly one that says, ‘Don’t eat me’.”

Since the publication of that study, other researchers determined the combined structure of CD47 and SIRPa together. Using this information, Discher’s group was able to computationally design the smallest sequence of amino acids that would act like CD47. This “minimal peptide” would have to fold and fit well enough into the receptor of SIRPa to serve as a valid passport.

After chemically synthesizing this minimal peptide, Discher’s team attached it to conventional nanoparticles that could be used in a variety of experiments.

“Now, anyone can make the peptide and put it on whatever they want,” Rodriguez said.

The research team’s experiments used a mouse model to demonstrate better imaging of tumors and as well as improved efficacy of an anti-cancer drug-delivery particle.

As this minimal peptide might one day be attached to a wide range of drug-delivery vehicles, the researchers also attached antibodies of the type that could be used in targeting cancer cells or other kinds of diseased tissue. Beyond a proof of concept for therapeutics, these antibodies also served to attract the macrophages’ attention and ensure the minimal peptide’s passport was being checked and approved.

“We’re showing that the peptide actually does inhibit the macrophage’s response,” Discher said. “We force the interaction and then overwhelm it.”

The test of this minimal peptide’s efficacy was in mice that were genetically modified so their mac[r]ophages had SIRPa receptors similar to the human version. The researchers injected two kinds of nanoparticles — ones carrying the peptide passport and ones without — and then measured how fast the mice’s immune systems cleared them.

“We used different fluorescent dyes on the two kinds of nanoparticles, so we could take blood samples every 10 minutes and measure how many particles of each kind were left using flow cytometry,” Rodriguez said. “We injected the two particles in a 1-to-1 ratio and 20-30 minutes later, there were up to four times as many particles with the peptide left.”

Even giving therapeutic nanoparticles an additional half-hour before they are eaten by macrophages could be a major boon for treatments. Such nanoparticles might need to make a few trips through the macrophage-heavy spleen and liver to find their targets, but they shouldn’t stay in the body indefinitely. Other combinations of exterior proteins might be appropriate for more permanent devices, such as pacemaker leads, enabling them to hide from the immune system for longer periods of time.

While more research is necessary before such applications become a reality, reducing the peptide down to a sequence of only a few amino acids was a critical step. The relative simplicity of this passport molecule to be more easily synthesized makes it a more attractive component for future therapeutics. …

A very interesting feature of this work is the computational identification of a small structure, in this case a peptide, that can substitute for a crucial part of the function of a large biological system (phagocytic cells to recognize non-self). We should probably expect to see this strategy often as nanomedicine evolves from predominantly biotechnology toward more machine-like advanced nanotechnology.
—James Lewis, PhD

Mesocosms. Credit: Benjamin Coleman

The advent of new technologies is typically followed by new government regulation, and in the absence of data, fear-based reactionism can have far too much influence on policy. Quality research studies on real risks and impacts of nanoscale technologies can help lead to legitimate scientific consensus and appropriate regulation.

Engineered nanoparticles draw particular attention, because the same unique properties that give rise to special utility may also give rise to special health and environmental risks.

To calibrate our responses to nanoparticle toxicology studies, it is important to note whether an experiment reasonably represents likely exposure scenarios and whether nanoscale size is in fact a contributing factor to observed effects.

Recently highlighted at Phys.org, researchers at Duke University are investigating environmental impacts of widely used silver nanoparticles by way of experiments that seek to represent real-world exposure levels.

Previous studies have involved high concentrations of the nanoparticles in a laboratory setting, which the researchers point out, doesn’t represent “real-world” conditions.

For their studies, the researchers created mesocosms, which are small, man-made structures containing different plants and microorganisms meant to represent the environment. They applied sludge with low doses of silver nanoparticles in some of the mesocosms, then compared plants and microorganisms from treated and untreated mesocosms after 50 days.

“We’re trying to come up with the data that can be used to help regulators determine the risks to the environment from silver nanoparticle exposures,” [said Benjamin Colman, a post-doctoral fellow in Duke's biology department and a member of the Center for the Environmental Implications of Nanotechnology (CEINT)].

“Our results show that silver nanoparticles in the biosolids, added at concentrations that would be expected, caused ecosystem-level impacts,” Colman said.

The researchers plan to continue to study longer-term effects of silver nanoparticles and to examine another ubiquitous nanoparticle – titanium dioxide.

Studies that do not elucidate the roles of different particle properties can still be of great benefit by drawing attention to studies that do, and by adding to the pool of reliable data. Most important is for researchers and the public alike to recognize the difference and to support policy that is sensible and appropriate.
-Posted by Stephanie C

(Credit: Courtesy of UCLA Engineering)

One of the most promising near-term applications of current nanotechnology is in targeted drug delivery to treat cancer. Despite the fact that a number of approaches based on very different areas of nanoscience have shown promise in delivering a wide variety of agents in different animal models of cancer, a number of challenges remain, principally involving the stability of the nanoparticles in the circulatory system, getting them into cancer cells, releasing the cargo to kill the cells, and the fact that cancer cells often have defenses against anti-cancer drugs. A core-shell nanoparticle has been cleverly adapted to deliver a particularly effective agent to where it is needed. A hat tip to ScienceDaily for reprinting this UCLA news release “Tiny capsule effectively kills cancer cells“:

Devising a method for more precise and less invasive treatment of cancer tumors, a team led by researchers from the UCLA Henry Samueli School of Engineering and Applied Science has developed a degradable nanoscale shell to carry proteins to cancer cells and stunt the growth of tumors without damaging healthy cells.

In a new study, published online Feb. 1 in the peer-reviewed journal Nano Today [abstract], a group led by Yi Tang, a professor of chemical and biomolecular engineering and a member of the California NanoSystems Institute at UCLA, reports developing tiny shells composed of a water-soluble polymer that safely deliver a protein complex to the nucleus of cancer cells to induce their death. The shells, which at about 100 nanometers are roughly half the size of the smallest bacterium, degrade harmlessly in non-cancerous cells.

The process does not present the risk of genetic mutation posed by gene therapies for cancer, or the risk to healthy cells caused by chemotherapy, which does not effectively discriminate between healthy and cancerous cells, Tang said.

“This approach is potentially a new way to treat cancer,” said Tang. “It is a difficult problem to deliver the protein if we don’t use this vehicle. This is a unique way to treat cancer cells and leave healthy cells untouched.”

The cell-destroying material, apoptin, is a protein complex derived from an anemia virus in birds. This protein cargo accumulates in the nucleus of cancer cells and signals to the cell to undergo programmed self-destruction.

The polymer shells are developed under mild physiological conditions so as not to alter the chemical structure of the proteins or cause them to clump, preserving their effectiveness on the cancer cells.

Tests done on human breast cancer cell lines in laboratory mice showed significant reduction in tumor growth.

“Delivering a large protein complex such as apoptin to the innermost compartment of tumor cells was a challenge, but the reversible polymer encapsulation strategy was very effective in protecting and escorting the cargo in its functional form,” said Muxun Zhao, lead author of the research and a graduate student in chemical and biomolecular engineering at UCLA.

Tang’s group continues to research ways of more precisely targeting tumors, prolonging the circulation time of the capsules and delivering other highly sought-after proteins to cancer cells.

There is nothing very interesting here from the standpoint of the eventual development of atomically precise manufacturing, but this work presents an excellent case for making the most of the current tools of nanotechnology and employing a deep knowledge of biotechnology and using imaging technology to see what happens inside of cells to develop a promising solution to a set of difficult and important problems.
—James Lewis, PhD

Basic manipulations of atoms and bonds by scanning probe have become familiar capabilities that follow similar protocols: the STM tip is precisely positioned relative to the molecule and then energy (tunneling current) is modulated to achieve particular operations, including scanning, translocation across the substrate surface, and making/breaking chemical bonds.  Going a tremendous step beyond the basics, Wilson Ho* of University of California, Irvine and colleagues recently reported the selective formation of two distinct types of chemical bonds between gold adatoms and the sulfurs of 1,4-bis(4’-(acetylthio)styryl)benzene molecules (converting Ac-S-DSB-S-Ac to Au-S-DSB-S-Au) on a NiAl(110) surface.

1,4-bis(4’-(acetylthio)styryl)benzene

First, selective dissociation was achieved by successively positioning the STM tip at precise positions above each acetyl group and increasing sample bias to cause bond rupture, leaving a substrate-stabilized S-DSB-S molecule.

Each sulfur atom retained a lone pair and an unpaired electron which were available for bonding. Individual gold atoms were then manipulated toward the sulfurs from specific directions that targeted a particular electron group, and a pulse of energy was supplied from the STM tip to trigger discreet bonding. Bonding via the unpaired electron produced a covalent bond, while bonding via the lone pair produced a coordinate bond.

In this stepwise manner, the team was able to selectively produce molecules containing a covalent bond between one S-Au pair and a coordinate bond between the other S-Au pair, as well as molecules containing two covalently bound S-Au pairs or two coordinate pairs, for a total of nine distinct DSB-2S-2Au complexes to date.

Notably, the spatial location of the electron groups around the sulfur atoms appears to follow the symmetry of the DSB framework. Dr. Wilson Ho explained that, “Once the symmetry of the DSB is imaged and determined, we can infer the electron groups on the exposed sulfurs. The Au atoms can then be manipulated to form different types of bonds depending on whether they attach to the lone pair or the unpaired electron of the S atoms.”

This remarkable use of directionality for selective bond formation serves to further illustrate the fundamental accessibility of machine-guided synthesis: orbital overlap and energy barriers are key, and manipulation approaches will be increasingly understood and exploited as molecular fabrication technologies continue to develop.
*Sincere thanks to Dr. Wilson Ho for generous communications regarding this work
-Posted by Stephanie C

Harmless bacteria could be re-engineered into microscopic factories that could, in addition to more immediate applications, perhaps provide components for more general molecular manufacturing systems. (credit Imperial College, London)

Synthetic biology and molecular manufacturing/productive nanosystems have in common the effort to rationally engineer systems to make and assemble parts for complex molecular machine systems. The effort in synthetic biology to design complex biological systems in a hierarchical architecture from well-characterized molecular parts is accelerating. A hat tip to ScienceDaily for reprinting this Imperial College news release “Discovery in synthetic biology a step closer to new industrial revolution“:

Scientists report that they have developed a method that cuts down the time it takes to make new ‘parts’ for microscopic biological factories from 2 days to only 6 hours.

The scientists, from Imperial College London, say their research brings them another step closer to a new kind of industrial revolution, where parts for these biological factories could be mass-produced. These factories have a wealth of applications including better drug delivery treatments for patients, enhancements in the way that minerals are mined from deep underground and advances in the production of biofuels.

Professor Paul Freemont, Co- Director of the Centre for Synthetic Biology and Innovation at Imperial College London and principal co-investigator of the study, which is published today in the journal Nucleic Acids Research [abstract, free full text PDF], says:

“Before the industrial revolution most items were made by hand, which meant that they were slower to manufacture, more expensive to produce and limited in number. We are at a similar juncture in synthetic biology, having to test and build each part from scratch, which is a long and slow process. We demonstrate in our study a new method that could help to rapidly scale up the production and testing of biological parts.”

Parts made up of DNA are re-engineered by scientists and put into cells to make biological factories. However, a major bottleneck in synthetic biology is the lack of parts from which to build new types of factories. To build parts using the current time-consuming method, scientists have to re-engineer DNA in a cell and observe how it works. If it functions according to their specifications, then the scientists store the part specifications in a catalogue.

Now, scientists from Imperial College London have devised a much quicker method that does away with the need for them to re-engineer a cell every time they want to make a new part. The team say their work could lead to vast new libraries of off-the-shelf components that could be used to build more sophisticated biological factories.

James Chappell, co-author of the study from the Centre for Synthetic Biology and Innovation at Imperial College London, says:

“One of the major goals in synthetic biology is to find a way to industrialise our processes so that we can mass produce these biological factories much in the same way that industries such as car manufacturers mass produce vehicles in a factory line. This could unlock the potential of this field of science and enable us to develop much more sophisticated devices that could be used to improve many facets of society. Excitingly, our research takes us one step closer to this reality, providing a rapid way of developing new parts.”

When a cell is re-engineered, the re-programmed DNA in the cell encodes a message that is conveyed by molecules called messenger ribonucleic acid (mRNA) to the cell’s production factories called ribosomes. The ribosomes translate the genetic information into a command that instructs the cell to perform functions. For example, scientists can already re-engineer a cell into an infection detector factory, which produces a protein that detects chemical signals from human pathogenic bacteria and changes colour to indicate their presence.

In the study, the Imperial researchers demonstrate for the first time that the same method can be achieved in a test tube outside of a cell. This involves extracting from cells the machinery that produces mRNA and proteins and providing the energy and building blocks to help them survive in test tubes. The team then add their re-programmed DNA to the solution and observe how it functions.

The advantage of this method is that scientists can develop litres of this cell-like environment so that multiple re-programmed DNA can be tested simultaneously, which speeds up the production process of parts.

The next stage of the research is to expand the types of parts and devices that can be developed using this method. They also are aiming to develop a method using robots to speed up and make the whole process automated.

Professor Richard Kitney, co- Director of the Centre for Synthetic Biology and Innovation at Imperial College London says: “Synthetic biology is seen by the British Government as having the potential to create new industries and jobs for the benefit of the UK economy. This work is part of a wider, major research programme within the Centre to develop technology that can be used across a range of industrial applications.”

The hope driving this research is that biological parts will transform industrial sectors like drug delivery and biofuels production into molecular manufacturing processes. Whether synthetic biology can eventually be made to contribute parts for nanofactories to implement more general molecular manufacturing using stronger, more rigid parts, remains to be seen.
—James Lewis, PhD

(Credit: Miriam Wilson)

Nature has evolved a partial system of molecular manufacturing. Although not capable of making arbitrarily complex, atomically precise structures that faithfully represent the immense diversity of chemically feasible structures, biology very early on evolved a method of assembling a wide range of molecular machines by linking particular types of subunit molecules together in a defined sequence. The structure that makes the most complex biological molecular machines is the ribosome, a complex of two subunits 20 to 30 nm in diameter and comprising several dozen RNA and protein molecules, which works with hundreds of other RNA and protein molecules to link amino acids together as specified by genetic information to make specific protein molecules. Scientists in the UK have succeeded in mimicking this basic process using a simple artificial molecular machine bearing no resemblance to the ribosome and an order of magnitude smaller in linear dimension than the ribosome. A hat tip to Phys.org for reprinting this University of Manchester news release “Molecular machine could hold key to more efficient manufacturing“:

An industrial revolution on a minute scale is taking place in laboratories at The University of Manchester with the development of a highly complex machine that mimics how molecules are made in nature.

The artificial molecular machine developed by Professor David Leigh FRS and his team in the School of Chemistry is the most advanced molecular machine of its type in the world. Its development has been published in the journal Science [abstract].

Professor Leigh explains: “The development of this machine which uses molecules to make molecules in a synthetic process is similar to the robotic assembly line in car plants. Such machines could ultimately lead to the process of making molecules becoming much more efficient and cost effective. This will benefit all sorts of manufacturing areas as many manmade products begin at a molecular level. For example, we’re currently modifying our machine to make drugs such as penicillin.” …

Professor Leigh’s machine is based on the ribosome. It features a functionalized nanometre-sized ring that moves along a molecular track, picking up building blocks located on the path and connecting them together in a specific order to synthesize the desired new molecule.

First the ring is threaded onto a molecular strand using copper ions to direct the assembly process. Then a “reactive arm” is attached to the rest of the machine and it starts to operate. The ring moves up and down the strand until its path is blocked by a bulky group. The reactive arm then detaches the obstruction from the track and passes it to another site on the machine, regenerating the active site on the arm. The ring is then free to move further along the strand until its path is obstructed by the next building block. This, in turn, is removed and passed to the elongation site on the ring, thus building up a new molecular structure on the ring. Once all the building blocks are removed from the track, the ring de-threads and the synthesis is over.

Professor Leigh says the current prototype is still far from being as efficient as the ribosome: “The ribosome can put together 20 building blocks a second until up to 150 are linked. So far we have only used our machine to link together 4 blocks and it takes 12 hours to connect each block. But you can massively parallel the assembly process: We are already using a million million million (10¹⁸) of these machines working in parallel in the laboratory to build molecules.”

Professor Leigh continues: “The next step is to start using the machine to make sophisticated molecules with more building blocks. The potential is for it to be able to make molecules that have never been seen before. They’re not made in nature and can’t be made synthetically because of the processes currently used. This is a very exciting possibility for the future.”

An animation illustrating how the molecular machine works is available on YouTube. Professor Leigh was the winner of the 2007 Foresight Institute Feynman Prize in Theory for the design and synthesis of artificial molecular machines.

The small molecular machine in this work is based upon a rotaxane molecule, in which a dumbbell-shaped molecule is threaded through a macrocycle. The dumbbell is constructed from two components: a “stopper”, and a “strand”, along which the amino acids to be added are linked. A chemical moiety that will link with the stopper defines the front of the strand. The order of the amino acid subunits in the peptide molecule to be manufactured is determined by the order in which the reactive building blocks are positioned along the molecular strand, with the first one nearest the front, and the last at the opposite end of the strand. Following the practice for solid state chemical synthesis of peptides pioneered by Bruce Merrifield half a century ago, the amino groups of the amino acids are protected with a chemical group that keeps them from reacting prematurely. The third component of the rotaxane is the macrocycle, which has a site at which the reactive arm of the molecular machine will be attached.

Three amino acids are linked to the strand, separated from each other by rigid spacers: phenylalanine at the front, then leucine, then alanine. As with conventional solid state peptide synthesis, the peptide will be synthesized from the C-terminal to the N-terminal end, the opposite of direction of synthesis on the ribosome. The rotaxane is assembled in a chemical reaction catalyzed by copper ions in which the macrocycle is guided over the front of the strand as the strand is linked to the stopper. The macrocycle is held in place, at the point where the stopper and strand join, by a bulky substituent on the outside of the stopper, which forms one end of the dumbbell, and by the first amino acid (phenylalanine) on the strand, which forms the other end.

A second chemical reaction added to the macrocycle the reactive arm responsible for manufacturing the peptide. The reactive arm is a tripeptide (GlyGlyCys) attached to the macrocycle by its C-terminal cysteine residue. The thiolate group of the cysteine and the N-terminus of the tripeptide are both blocked by chemical protective groups to render them non-reactive. The machine is activated by acid-catalyzed removal of the protective groups from the thiolate, from the amino terminus of the reactive arm, and from the amino groups of the three amino acids on the strand.

The liberated thiolate is now able to attack the bond linking the C-terminus of phenylalanine to the strand (the other two amino acids on the strand are too distant to react), thus freeing phenylalanine from the strand, linking it to the N-terminus of reactive arm tripeptide, and regenerating the thiolate. The reactive arm is now longer by one amino acid at its N-terminus, and the macrocycle is now free to slide along the strand until it encounters the second amino acid. The authors explain that the glycylglycine was added to the N-terminus of the cysteine residue because the thiolate and the N-terminus of the cysteine are too close to each other for the phenylalanine released from the strand to be added to the N-terminus of the cysteine. The peptide formed is thus, from the N- toward the C-terminus, PheGlyGlyCys.

The process repeats twice more until all three amino acids have been removed from the strand and attached to the N-terminus of the peptide growing on the reactive arm. With no amino acids left on the strand, the macrocycle is free to slide off the end, bearing the newly formed hexapeptide: AlaLeuPheGlyGlyCys.

Clearly this novel artificial molecular machine was able to mimic essential features of ribosomal protein synthesis, as intended. Once the reaction was initiated, the synthesis was completed without further manipulation. As noted by the authors, major limitations include the very slow rate of synthesis, the destruction of the template after the synthesis is completed, and that the size of the peptide produced my be limited by the nature of the transition state of the machine during each cycle. Nevertheless, this work has demonstrated:

… that relatively small, highly modular, artificial molecular machines can be designed to autonomously perform iterative tasks in synthesis. The principles employed in the design and operation of 1 should be broadly applicable to other types of monomer and chemical reactions …

From the standpoint of the eventual development of molecular manufacturing, the important question will be whether or not this type of molecular machine can be extended to do more general positional control of chemical synthesis, or will it be limited to linking monomers together to form various types of folding polymers?
—James Lewis, PhD