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Expanded DNA alphabet provides more options for nanotechnology

Floyd E. Romesberg, associate professor at Scripps Research (Credit: The Scripps Research Institute)

Long-time readers of Nanodot may remember the section of Chapter 15 of Nanosystems in which Drexler explores options for producing easier to design proteins for the protein engineering path toward atomically precise manufacturing by incorporating specially chosen amino acids in addition to the 20 genetically encoded amino acids. Back in 1992 the only option for incorporating unnatural amino acids into proteins was Merrifield solid phase peptide synthesis, using the methods of organic chemistry rather than biological systems. However, this becomes problematic and expensive for longer chains. Consequently, finding ways to expand the repertoire of biologically encoded amino acids would be quite useful. One way to accomplish this goal would be to expand the DNA ‘alphabet’ from two to three base pairs (that is, from four to six ‘letters’). We noted progress in this direction back in February of 2008 when Floyd Romesberg, at the Scripps Research Institute, La Jolla, California created two artificial DNA letters that were accurately and efficiently replicated by a natural enzyme. In September of 2011 we noted a different approach taken by a team at the Salk Institute that keeps the current DNA alphabet but alters one three-letter word to mean an unnatural amino acid, increasing the amino acid repertoire by one. We noted in June of 2012 that continued work by Romesberg had revealed how the new base pair was efficiently replicated in the test tube by a natural enzyme. In a major advance, Romesberg and his collaborators have engineered a living organism to stably propagate the expanded genetic alphabet. The research was published in Nature [abstract] and was nicely described in a news article in Science by Robert F. Service “Designer Microbes Expand Life’s Genetic Alphabet“:

From bacteria to basketball players, all life as we know it encodes genetic information using two pairs of DNA letters. Not anymore. Now, along with the double helix’s two natural pairs—A bound to T and G bound to C—a bacterium growing in a California lab can incorporate and copy a third, artificial pair of letters. For now, the artificial bases—call them X and Y—don’t code for anything, unlike natural DNA base pairs, which in various combinations code for the 20 different amino acids that make up proteins. But the newly expanded genetic code opens the door for synthetic biologists to create microbes capable of building their proteins out of as many as 172 different amino acids, both natural and artificial—a potential boon to drug and materials developers. …

Getting live bacteria to replicate altered DNA was another challenge entirely. The bacteria would need either to synthesize the new genetic letters themselves or to import them from the surrounding culture medium. In algae, Romesberg and his colleagues identified a protein that grabs nucleotide bases and pulls them into the cell. They spliced the gene for this transporter protein into Escherichia coli bacteria and found it enabled the bacteria to pull in presynthesized X and Y bases as well. The team had also engineered their E. coli to harbor small rings of DNA called plasmids carrying X-Y pairs. When the bacteria copied those plasmids, they used the newly imported X and Y bases—yet the engineered cells grew just as well as their native cousins.

Next, Romesberg says he hopes to use his expanded genetic alphabet to create designer proteins. Scripps biochemist Peter Schultz and others have already engineered bacteria to build proteins with dozens of amino acids beyond nature’s standard 20. But those experiments use natural DNA to code for unnatural amino acids. The newly expanded genetic alphabet, Thyer says, should yield a vastly more diverse menu of proteins with a wide variety of new chemical functions, such as medicines better able to survive in the body and protein-based materials that assemble themselves. Romesberg says forays into that new world of proteins are already under way.

An article in The Scientist by Kate Yandell provides additional details “Augmenting the Genetic Alphabet“:

… “This is the first paper to show the possibility that living organisms can have really artificial DNA with [an] expanded genetic alphabet,” Ichiro Hirao, a synthetic biologist at the RIKEN Center for Life Science Technologies in Japan, wrote in an e-mail to The Scientist. Hirao was not involved in the study, but is also working to incorporate synthetic base pairs into living organisms. …

Certain intracellular bacteria and organelles within algae do not create their own nucleotides. Rather, they import key nucleoside triphosphates from their environments via membrane transport proteins. Romesberg and colleagues tested various triphosphate transporters and eventually found that one, from a plastid in a diatom, could import synthetic triphosphates into E. coli. They added a gene for the transporter to E. coli on a plasmid and optimized the cells’ food so that they would ingest the triphosphates.

Romesberg thought that multiple additional challenges remained, including getting the cell’s DNA polymerases to recognize and replicate the base pairs and preventing the cell’s DNA repair mechanisms from recognizing the novel nucleotides as aberrant. But, much to the team’s surprise, the unnatural plasmids immediately began to replicate. …

Romesberg and his colleagues are now working to get RNA polymerases to transcribe the novel DNA into mRNAs. Then they hope to engineer cells to transcribe tRNAs that will read novel codons and translate novel amino acid sequences into proteins. Romesberg suggested that synthetic biologists will eventually build proteins with both natural amino acids currently not used in proteins and amino acids synthesized in the lab. He added that Synthorx, a company created based on his work, and of which he was scientific founder, aims to ramp up the production of synthetic triphosphates. …

From a Scripps news release “Scripps Research Institute Scientists Create First Living Organism that Transmits Added Letters in DNA ‘Alphabet’“:

Scientists at The Scripps Research Institute (TSRI) have engineered a bacterium whose genetic material includes an added pair of DNA “letters,” or bases, not found in nature. The cells of this unique bacterium can replicate the unnatural DNA bases more or less normally, for as long as the molecular building blocks are supplied.

“Life on Earth in all its diversity is encoded by only two pairs of DNA bases, A-T and C-G, and what we’ve made is an organism that stably contains those two plus a third, unnatural pair of bases,” said TSRI Associate Professor Floyd E. Romesberg, who led the research team. “This shows that other solutions to storing information are possible and, of course, takes us closer to an expanded-DNA biology that will have many exciting applications—from new medicines to new kinds of nanotechnology.” …

Romesberg and his laboratory have been working since the late 1990s to find pairs of molecules that could serve as new, functional DNA bases—and, in principle, could code for proteins and organisms that have never existed before.

The task hasn’t been a simple one. Any functional new pair of DNA bases would have to bind with an affinity comparable to that of the natural nucleoside base-pairs adenine–thymine and cytosine–guanine. Such new bases also would have to line up stably alongside the natural bases in a zipper-like stretch of DNA. They would be required to unzip and re-zip smoothly when worked on by natural polymerase enzymes during DNA replication and transcription into RNA. And somehow these nucleoside interlopers would have to avoid being attacked and removed by natural DNA-repair mechanisms. …

The next step will be to demonstrate the in-cell transcription of the new, expanded-alphabet DNA into the RNA that feeds the protein-making machinery of cells. “In principle, we could encode new proteins made from new, unnatural amino acids—which would give us greater power than ever to tailor protein therapeutics and diagnostics and laboratory reagents to have desired functions,” Romesberg said. “Other applications, such as nanomaterials, are also possible.”

Several of the above reports commented that the fact that the two artificial nucleotides have to be supplied to the organism—they cannot be manufactured by the organism—is a strong plus from a safety standpoint. There is no chance of the organisms escaping and replicating in an uncontrolled fashion. Beyond the immediate application of designing better protein folds, the expanded genetic alphabet opens a path for synthetic biology to become a much larger playground for developing very complex systems of molecular machines. Where it will lead seems impossible to predict.
—James Lewis, PhD

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Nanotechnology to provide efficient, inexpensive water desalination

Credit: O’Hern, S. C. et al./Nano Letters

Another area in which incremental nanotechnology is poised to make a major contribution to human welfare through increasing control of the atomic structure of bulk materials is Supplying Clean Water Globally. Two recent reports use slightly different chemistries to achieve similar results: water desalination and purification.

KurzweilAI describes research in which gallium ions and oxidative etching were used to create sub-nanometer diameter holes in single layer graphene membranes “Selective nanopores in graphene dramatically improve desalination and purification“:

A team of researchers at MIT, Oak Ridge National Laboratory, and in Saudi Arabia succeeded in creating subnanoscale pores in a sheet of graphene, a development that could lead to ultrathin filters for improved desalination or water purification. Their findings are published in the journal Nano Letters. [abstract]

The new work, led by graduate student Sean O’Hern and associate professor of mechanical engineering Rohit Karnik, is the first step toward actual production of such a graphene filter.

Making these minuscule holes in graphene — a hexagonal array of carbon atoms, like atomic-scale chicken wire — occurs in a two-stage process. First, the graphene is bombarded with gallium ions, which disrupt the carbon bonds. Then, the graphene is etched with an oxidizing solution that reacts strongly with the disrupted bonds — producing a hole at each spot where the gallium ions struck. By controlling how long the graphene sheet is left in the oxidizing solution, the MIT researchers can control the average size of the pores. …

The permeability of such graphene filters, according to computer simulations, could be 50 times greater than that of conventional membranes, as demonstrated earlier by a team of MIT researchers led by graduate student David Cohen-Tanugi of the Department of Materials Science and Engineering. But producing such filters with controlled pore sizes has remained a challenge. The new work, O’Hern says, demonstrates a method for actually producing such material with dense concentrations of nanometer-scale holes over large areas. …

With this technique, the researchers were able to control the filtration properties of a single, centimeter-sized sheet of graphene: Without etching, no salt flowed through the defects formed by gallium ions. With just a little etching, the membranes started allowing positive salt ions to flow through. With further etching, the membranes allowed both positive and negative salt ions to flow through, but blocked the flow of larger organic molecules. With even more etching, the pores were large enough to allow everything to go through.…

In similar research, also reported by KurzweilAI, a micrometer-thick laminate of graphene oxide hosts a network of nanocapillaries that blocks all solutes with hydrated radii greater than 0.45 nm “New multilayer graphene structure allows ‘ultraprecise,’ ‘ultrafast’ water filtering“:

University of Manchester researchers have taken another key step toward a seawater filter: they’ve developed one-atom-wide graphene-oxide (GO) capillaries by building multilayer GO membranes (laminates).

As described in Science [abstract], these new laminates allow for “ultraprecise” selection of molecules that can go through the filter and “ultrafast” flow of water.

The new GO filters have an “astonishingly” accurate mesh that allows them to distinguish between atomic species that are only a few percent different in size. The filters block passage of ions (charged atoms) that are larger than 9 angstroms (.9 nanometers).

“The water filtration is as fast and as precise as one could possibly hope for such narrow capillaries,” said study co-author Rahul Nair. “Now we want to control the graphene mesh size and reduce it below nine Angstroms to filter out even the smallest salts like in seawater. Our work shows that it is possible.”

“Our ultimate goal is to make a filter device that allows a glass of drinkable water made from seawater after a few minutes of hand pumping,” added study co-author Irina Grigorieva. “We are not there yet but this is no longer science fiction.”

As the remainder of the article explains, graphene oxide is hydrophobic (repels water) but a water layer only one molecule thick will flow rapidly through narrow capillaries of graphene. It turns out that a quick and easy way to make graphene oxide capillaries with a one-atom-wide diameter is to pile layers of graphene oxide together, forming a very strong laminate a micrometer thick. These laminates are vacuum-tight when dry, but when immersed in water a network of nanocapillaries forms that accept only species smaller than 0.9 nm in diameter, and at rates thousands of times faster than allowed by diffusion. The diameter of a water molecule is about 0.28 nm. The diameter of a sodium ion is about 0.23 nm and of a chloride ion about 0.33 nm, so salt as well as water will pass through the current laminates, although larger molecules would be excluded. The authors seem confident that they can make the capillaries small enough to exclude salt ions but pass water molecules.

The quick and inexpensive separation of pure water from seawater or other sources would be easiest with a one-atom-thick membrane perforated with large numbers of precisely placed atomically precise pores just large enough to efficiently pass water molecules and block all other atoms, molecules, and ions. In the absence of a method for atomically precise manufacturing of such a material, the above two research collaborations succeeded in using current nanotechnology of bulk materials to place large numbers of nanometer-scale pores controlled to near atomic precision in bulk nanoscale materials. Either or both of these approaches, or some similar approach, may take us a long way to meeting all or most of our ultrafiltration and purification needs, but when we develop atomically precise manufacturing we will have simpler and easier access to filters designed for the exact purpose needed.
—James Lewis, PhD

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Nanotechnology to provide better solar cells, optical devices

Electron microscope picture of wurtzite GaAs/AlGaAs core-shell nanowires. Credit: Dr. Dheeraj Dasa and Prof. Helge Weman, NTNU

While we work for the eventual development of a nanotechnology that transforms human life via atomically precise manufacturing, the partial control of the configuration of atoms in important materials that is afforded by current nanotechnology promises great near-term advantages. A decade ago, Foresight focused on progress in nanotechnology to meet six major challenges faced by humanity. Although we haven’t said as much the past several years about these challenges (except for #3, Improving Health and Longevity), recent progress promises great contributions to the other challenges as well. Challenge #1, Providing Renewable Clean Energy, appears soon to profit from advances in controlling the atomic configuration of gallium arsenide nanowires. Patrick Cox’s Tech Digest reports on “Building a Better Solar Cell One Atom at a Time“. Citing work by researchers at the Norwegian University of Science and Technology working with IBM engineers to grow gallium arsenide nanowires on graphene, he concludes:

… With a better understanding of how, atom by atom, a panel’s composition could be manipulated to achieve maximum output, solar-panel technology of the future promises to become lighter and more portable, as well as easier to manufacture and maintain. …

A hat tip to ScienceDaily for providing more details by reprinting news published by the Norwegian University of Science and Technology “Better Solar Cells, Better LED Light And Vast Optical Possibilities“:

Changes at the atom level in nanowires offer vast possibilities for improvement of solar cells and LED light. NTNU-researchers have discovered that by tuning a small strain on single nanowires they can become more effective in LEDs and solar cells.

NTNU researchers Dheeraj Dasa and Helge Weman have, in cooperation with IBM, discovered that gallium arsenide can be tuned with a small strain to function efficiently as a single light-emitting diode or a photodetector. This is facilitated by the special hexagonal crystal structure, referred to as wurtzite, which the NTNU researchers have succeeded in growing in the MBE lab at NTNU. The results were published in Nature Communications [abstract].

… By altering the crystal structure in a substance, i.e. changing the positions of the atoms, the substance can gain entirely new properties. The NTNU researchers discovered how to alter the crystal structure in nanowires made of gallium arsenide and other semiconductors.

With that, the foundation was laid for more efficient solar cells and LEDs.

“Our discovery was that we could manipulate the structure, atom by atom. We were able to manipulate the atoms and alter the crystal structure during the growth of the nanowires. This opened up for vast new possibilities. We were among the first in the world who were able to create a new gallium arsenide material with a different crystal structure,” says Helge Weman at the Department of Electronics and Telecommunications.

… The next big news came in 2012. At that point, the researchers had managed to make semiconductor nanowires grow on the super-material graphene. Graphene is the thinnest and strongest material ever made. This discovery was described as a revolution in solar cell and LED component development.

… The research group has received a lot of international attention for the graphene method. Helge Weman and his NTNU co-founders Bjørn-Ove Fimland and Dong-Chul Kim have established the company CrayoNano AS, working with a patented invention that grows semiconductor nanowires on graphene. The method is called molecular beam epitaxy (MBE), and the hybrid material has good electric and optical properties.

“We are showing how to use graphene to make much more effective and flexible electronic products, initially solar cells and white light-emitting diodes (LED). The future holds much more advanced applications,” says Weman.

… The last couple of years the research group has, among other things, studied the unique hexagonal crystal structure in the GaAs nanowires.

“In cooperation with IBM, we have now discovered that if we stretch these nanowires, they function quite well as light-emitting diodes. Also, if we press the nanowires, they work quite well as photodetectors. This is facilitated by the hexagonal crystal structure, called wurtzite. It makes it easier for us to change the structure to optimise the optical effect for different applications.

“It also gives us a much better understanding, allowing us to design the nanowires with a built-in compressive stress, for example to make them more effective in a solar cell. This can for instance be used to develop different pressure sensors, or to harvest electric energy when the nanowires are bent,” Weman explains.

Because of this new ability to manipulate the nanowires’ crystal structure, it is possible to create highly effective solar cells that produce a higher electric power. Also, the fact that CrayoNano now can grow nanowires on super-light, strong and flexible graphene, allows production of very flexible and lightweight solar cells.

The CrayoNano group will now also start growing gallium nitride nanowires for use in white light-emitting diodes.

“One of our objectives is to create gallium nitride nanowires in a newly installed MBE machine at NTNU to create light-emitting diodes with better optical properties — and grow them on graphene to make them flexible, lightweight and strong.”

This work illustrates beautifully the practical benefits from increasing ability to control the configuration of atoms in materials. This work uses currently available tools to control the atomic configuration of bulk materials. Atomically precise manufacturing, when it is developed, will allow specifying different atomic configurations to produce, if needed, nanometer scale complexity throughout a microscale or macroscale object to manufacture enormously complex systems of materials, devices, and molecular machines.
—James Lewis, PhD

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A bird’s-eye view of half a century of nanotechnology

The Foresight Institute was founded on the vision of nanotechnology put forward by Eric Drexler in his 1986 popular science work Engines of Creation, and clarified in his 1992 technical study Nanosystems. For the flavor of thinking about nanotechnology around 1987, see here and here. We’ve mentioned Drexler’s new book Radical Abundance here on Nanodot several times during the past year, for example here. Over at The Freeman, Phil Bowermaster discusses Radical Abundance in the context of the conversation about nanotechnology over the past 28 years — “The Reluctant Visionary“:

In 1959, Richard Feynman delivered a lecture with the provocative title “There’s Plenty of Room at the Bottom.” Speaking at a meeting of the American Physical Society at Caltech, the Nobel-laureate-to-be speculated about the possibility of manipulating matter at the atomic level via exquisitely small machines. Would it be possible, Feynman asked, for such machinery to configure atoms themselves, producing atomically precise outputs? Might we one day have billions of submicroscopic factories working in parallel to produce anything and everything we need?

It was a profound and exciting idea, and yet one that received very little serious attention in the years that followed, until an MIT student named K. Eric Drexler took up the cause in the 1980s. Working within Marvin Minsky’s MIT Media Lab, Drexler earned a Ph.D. in molecular nanotechnology—the first such degree ever awarded anywhere. Along the way he wrote the bestselling Engines of Creation (1986), which outlined his vision of nanotechnology for non-technical audiences, and the technical treatise Nanosystems (1991) [Note: Drexler’s PhD thesis, upon which Nanosystems is based, was completed in 1991; Nanosystems was published in 1992], which got into the nuts and bolts of nanotech.

Engines of Creation kicked off a worldwide nanotechnology craze. Corporations and universities began sponsoring research. Governments formed committees to develop technology roadmaps. Speculation in the media and popular culture grew ever wilder and more colorful, promoting images of tiny robots that could keep our clothes stain-free and our arteries unclogged, provided they didn’t go into an unstoppable feeding frenzy and reduce the entire world to a quivering mass of goo. Along with this buzz grew skepticism as to when and if we would ever see such technology, and whether molecular nanotechnology as described by Drexler was even possible. …

Bowermaster’s first three paragraphs succinctly summarize the public reaction to nanotechnology up through 2003. The debates at that time between Drexler and other advocates for advanced nanotechnology, on one side, and Nobel laureate and nanotechnology pioneer Richard Smalley and other skeptics on the other side, were extensively covered by Foresight Update (see for example, here, here, and here). In a paper published in 2004 [abstract, PDF courtesy of Center for Responsible Nanotechnology] Chris Phoenix and Eric Drexler put to rest the idea of the accidental creation of an all-devouring goo. Also in 2004 Drexler published an article in Bulletin Of Science, Technology & Society [abstract, PDF courtesy of Dr. Drexler] revealing the foundations of the debates and of skepticism about advanced nanotechnology, which at that time was generally referred to as molecular manufacturing.

In the remainder of the article Bowermaster describes the vision of atomically precise manufacturing (APM) and its revolutionary implications presented in Radical Abundance. The toll of the previous decade’s battles with Smalley and others is proposed as the reason that Radical Abundance eschews descriptions of the most exciting future applications of atomically precise manufacturing in favor of very sober arguments as to why the advent of APM is likely, and why serious conversations are needed now.

Clearly Drexler wants serious conversations unencumbered by sensational speculations (whether reasonably based or not) to prepare world society for whatever shocks and dislocations the advent of APM will bring. Bowermaster, however, suspects that real dialog will not begin until we are overwhelmed by those shocks. Founded on the vision put forth in Engines of Creation, Foresight also advocates serious preparations to obtain the benefits of APM and other transformative emerging technologies while avoiding the potential dangers. An initial step in charting the path to APM was the release in 2007 of the Technology Roadmap for Productive Nanosystems. Furthering the conversation on APM was the goal of The 2013 Foresight Technical Conference—Illuminating Atomic Precision. The 2014 Integration Conference looked at progress across a range of application areas in integrating current molecular and nanoscale elements into more complex micro- and macro-systems. Although not all current nanotechnologies lie on the path to APM (Drexler argues very few do), useful and profitable applications of current nanoscale science and technology may complement the physics-based arguments of Radical Abundance in drawing attention to the potential and significance of APM. It is important to discern what is on the direct path to APM and what plays supporting roles. Nevertheless, both types of nanotechnology deserve support, and progress in one area can accelerate progress in another.
—James Lewis, PhD

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To fight inflammation nanoparticles turn ‘naughty’ neutrophils into ‘nice’ neutrophils

Bottom right shows green-labeled neutrophils with red-labeled nanoparticles inside, which appear yellow. Credit University of Illinois at Chicago

A core advantage of nanomedicine is that appropriately designed nanoparticles can be targeted to deliver drugs to a very specific subset of cells in the body. An elegant example of specificity targets immune cells called neutrophils that are actively involved in damaging vascular inflammation while sparing neutrophils in circulation that are needed for other functions. A hat tip to Science Daily for reprinting this University of Illinois at Chicago news release written by Sharon Parmet “Nanoparticles target anti-inflammatory drugs where needed“:

Researchers at the University of Illinois at Chicago have developed a system for precisely delivering anti-inflammatory drugs to immune cells gone out of control, while sparing their well-behaved counterparts. Their findings were published online Feb. 23 in Nature Nanotechnology [abstract].

The system uses nanoparticles made of tiny bits of protein designed to bind to unique receptors found only on neutrophils, a type of immune cell engaged in detrimental acute and chronic inflammatory responses.

In a normal immune response, neutrophils circulating in the blood respond to signals given off by injured or damaged blood vessels and begin to accumulate at the injury, where they engulf bacteria or debris from injured tissue that might cause infection. In chronic inflammation, neutrophils can pile up at the site of injury, sticking to the blood vessel walls and to each other and contributing to tissue damage.

Adhesion of neutrophils to blood vessel walls is a major factor in acute lung injury, where it can impair the exchange of gases between the lungs and blood, leading to severe breathing problems. If untreated, the disease has a 50 percent mortality rate in intensive care units.

Corticosteroids and non-steroidal anti-inflammatory drugs used to treat inflammatory diseases are “blunt instruments that affect the whole body and carry some significant side effects,” says Asrar B. Malik, the Schweppe Family Distinguished Professor and head of pharmacology in the UIC College of Medicine, who is lead author of the paper.

Neutrophils that are stuck to blood vessels or clumped together have unique receptors on their surface that circulating neutrophils lack. Malik and his colleagues designed a nanoparticle to take advantage by embedding it with an anti-inflammatory drug. The nanoparticles bind to the receptors, and the neutrophils internalize the nanoparticle. Once inside, the anti-inflammatory drug works to “unzip” the neutrophil and allow it to re-enter the bloodstream.

“The nanoparticle is very much like a Trojan horse,” Malik said. “It binds to a receptor found only on these activated, sticky neutrophils, and the cell automatically engulfs whatever binds there. Because circulating neutrophils lack these receptors, the system is incredibly precise and targets only those immune cells that are actively contributing to inflammatory disease.”

Malik, along with research assistant professor Zhenjia Wang and assistant professor Jaehyung Cho, used intra-vital microscopy to follow nanoparticles in real-time in mice with induced vascular inflammation. The nanoparticles were labeled with a fluorescent dye, and could be seen binding to and entering neutrophils clustered together on the inner walls of capillaries, but not binding to freely circulating neutrophils. If the researchers attached a drug called piceatannol, which interferes with cell-cell adhesion, to the nanoparticles, they observed that clusters of neutrophils that took up the particles detached from each other and from the blood vessel wall. The cells were in effect neutralized and could no longer contribute to inflammation at the site of an injury.

The findings, Malik said, “show that nanoparticles can be used to deliver drugs in a highly targeted, specific fashion to activated immune cells and could be designed to treat a broad range of inflammatory diseases.”

Nanomedicine will advance hand-in-hand with biotechnology. As more is learned about proteins that are specifically displayed on cell surfaces in conjunction with disease, there will be more opportunities to design nanoparticles to target diseased cells.
—James Lewis, PhD

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Novel nanoparticle efficiently silences gene expression in liver cells

MIT engineers designed nanoparticles that can deliver short strands of RNA (green) into cells (nuclei are stained blue). Image credit: Gaurav Sahay, Yizhou Dong, and Omid Veiseh

One of the most promising weapons in the arsenal of today’s nanomedicine is to use specially designed nanoparticles to deliver siRNA to specific cells to exploit the power of RNA interference to silence the expression of specific genes. We have cited here progress in using various types of nanoparticles with some success in animal models of different diseases. A novel approach that combines systematic chemical modification of lipopeptides with inspiration provided by natural cholesterol-carrying particles appears close to clinical trials. A hat tip to ScienceDaily for reprinting this MIT news release written by Anne Trafton “Better RNA interference, inspired by nature“:

Inspired by tiny particles that carry cholesterol through the body, MIT chemical engineers have designed nanoparticles that can deliver snippets of genetic material that turn off disease-causing genes.

This approach, known as RNA interference (RNAi), holds great promise for treating cancer and other diseases. However, delivering enough RNA to treat the diseased tissue, while avoiding side effects in the rest of the body, has proven difficult.

The new MIT particles, which encase short strands of RNA within a sphere of fatty molecules and proteins, silence target genes in the liver more efficiently than any previous delivery system, the researchers found in a study of mice.

“What we’re excited about is how it only takes a very small amount of RNA to cause gene knockdown in the whole liver. The effect is specific to the liver — we get no effect in other tissues where you don’t want it,” says Daniel Anderson, the Samuel A. Goldblith Associate Professor of Chemical Engineering and a member of MIT’s Koch Institute for Integrative Cancer Research.

Anderson is senior author of a paper describing the particles in the Proceedings of the National Academy of Sciences the week of Feb. 10. Robert Langer, the David H. Koch Institute Professor at MIT, is also an author.

The research team, which included scientists from Alnylam Pharmaceuticals, also found that the nanoparticles could powerfully silence genes in nonhuman primates. The technology has been licensed to a company for commercial development. …

In previous studies, Anderson and Langer showed they could block multiple genes with small doses of siRNA by wrapping the RNA in fatlike molecules called lipidoids. In their latest work, the researchers set out to improve upon these particles, making them more efficient, more selective, and safer, says Yizhou Dong, a postdoc at the Koch Institute and lead author of the paper.

“We really wanted to develop materials for clinical use in the future,” he says. “That’s our ultimate goal for the material to achieve.”

The design inspiration for the new particles came from the natural world — specifically, small particles known as lipoproteins, which transport cholesterol and other fatty molecules throughout the body.

Like lipoprotein nanoparticles, the MIT team’s new lipopeptide particles are spheres whose outer membranes are composed of long chains with a fatty lipid tail that faces into the particle. In the new particles, the head of the chain, which faces outward, is an amino acid (the building blocks of proteins). Strands of siRNA are carried inside the sphere, surrounded by more lipopeptide molecules. Molecules of cholesterol embedded in the membrane and an outer coating of the polymer PEG help to stabilize the structure.

The researchers tuned the particles’ chemical properties, which determine their behavior, by varying the amino acids included in the particles. There are 21 amino acids found in multicellular organisms; the researchers created about 60 lipopeptide particles, each containing a different amino acid linked with one of three chemical groups — an acrylate, an aldehyde, or an epoxide. These groups also contribute to the particles’ behavior. …

The researchers then tested the particles’ ability to shut off the gene for a blood clotting protein called Factor VII, which is produced in the liver by cells called hepatocytes. Measuring Factor VII levels in the bloodstream reveals how effective the siRNA silencing is.

In that initial screen, the most efficient particle contained the amino acid lysine linked to an epoxide, so the researchers created an additional 43 nanoparticles similar to that one, for further testing. The best of these compounds, known as cKK-E12, achieved gene silencing five times more efficiently than that achieved with any previous siRNA delivery vehicle.

In a separate experiment, the researchers delivered siRNA to block a tumor suppressor gene that is expressed in all body tissues. They found that siRNA delivery was very specific to the liver, which should minimize the risk of off-target side effects.

“That’s important because we don’t want the material to silence all the targets in the human body,” Dong says. “If we want to treat patients with liver disease, we only want to silence targets in the liver, not other cell types.”

In tests in nonhuman primates, the researchers found that the particles could effectively silence a gene called TTR (transthyretin), which has been implicated in diseases including senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy.

The MIT team is now trying to learn more about how the particles behave and what happens to them once they are injected, in hopes of further improving the particles’ performance. They are also working on nanoparticles that target organs other than the liver, which is more challenging because the liver is a natural destination for foreign material filtered out of the blood.

The efficient and specific delivery of siRNA molecules is a long-standing problem. It is perhaps surprising that such large improvements could be made by trial and error chemical modifications of their lipopeptides. There is as yet no indication whether this approach will be useful for targeting organs other than the liver, but the degree of silencing in liver bodes well for clinical trials targeted to liver diseases.
—James Lewis, PhD

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Physicists suppress ‘stiction’ force that bedevils microscale machinery

Credit: Intravaia et al.

Whether or not MEMS (microelectromechanical systems) technology has use as a development path toward productive nanosystems, or atomically precise manufacturing, is unclear (see for example this series of posts on the Feynman Path by J. Storrs Hall), the problem of stiction in microscale mechanical systems has been used as a canard to criticize proposals for mechanical molecular machine systems. (For why this criticism is unfounded, see section 6.3.7 of Kinematic Self-Replicating Machines.) Nevertheless, MEMS is in its own right a very useful technology so it is gratifying to see that a solution to the stiction problem may be in sight. A hat tip to Dale Amon for pointing to this physics archive blog article “US Nuclear Weapons Laboratory Discovers How to Suppress the Casimir Force“:

The Casimir effect causes microscopic machines to stick fast. Now physicists have successfully tested a way to suppress this force

The Casimir effect is a strange and mysterious force that operates on the tiniest scales. It pushes together small metal objects when they are separated by a tiny distance.

That’s a problem because engineers are increasingly interested in building tiny machines with parts that move against each other on precisely the scale. For some years now, they’ve been thwarted by a problem called stiction in which the tiny cogs, gears and other parts in these machines stick together so tightly that the device stops working.

The culprit in these strange stiction events is often the Casimir effect. But since it is poorly understood, physicists and engineers have never known how to prevent it.

That looks set to change thanks to the work of Francesco Intravaia at Los Alamos National Laboratory in New Mexico and a few pals who have discovered a way to reduce this force and showed that it works for the first time. …

The research paper “Strong Casimir force reduction through metallic surface nanostructuring” is available at arxiv.org. The authors conclude that despite their successes achieved here, a full numerical analysis of the complexities of stiction in MEMS “remains an open problem.” Fortunately we already know that this does not have to be problem in a properly designed molecular machine system, even if implemented with diamondoid parts fashioned as nanoscale versions of macroscale machine parts.
—James Lewis, PhD

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US government report highlights flaws in US nanotechnology effort

Credit: GAO adapted from Executive Office of the President

Here at Nanodot we often report on basic research that may lie on the path to atomically precise manufacturing, and we also frequently report on nanoscale science and technology research that promises near-term revolutionary developments in medicine, computation, energy and other application areas, but we seldom have anything to say about the transition from research to commercial production. The United States Government Accountability Office (GAO) is worried about this same lack, and has identified an important nanotechnology policy gap. Last month Business Insider Australia reported “A New Report Warns That America May Lose The Nanotechnology Race“:

VACUUM TUBES, semiconductors and the internet have changed how we live; now nanotechnology promises a similar revolution. Nanocoatings that make it impossible for liquid to even touch a treated surface are transforming material science. Carbon nanotubes can help artificial muscles behave like the real thing, while nanoscale drug delivery can target cancer cells with deadly accuracy. Concrete infused with nanofibres can be self-sensing, enabling roads and bridges to be monitored remotely for structural weakness or traffic volumes. …

It is this breadth of nanotechnology’s potential that makes it vital to America’s future competitiveness. Congressman Lamar Smith, chairman of the House Committee on Science, Space, and Technology, believes that American dominance in the field has enormous economic potential and the ability to create new jobs: “it’s a game-changer that could transform and improve Americans’ daily lives in ways we can’t foresee,” he says.

On any measure — patents, private and government-sector investment, academic activity — America has so far been a leader in nanotechnology research and, to a lesser extent, development. …

So why is the United States Government Accountability Office (GAO), an independent agency that works for Congress and scrutinises how the federal government spends taxpayer dollars, now fretting that America may lose the nanotechnology race? In a new report on nanotechnology manufacturing (or nanomanufacturing) released today and prepared for Congressman Smith’s committee, the GAO finds flaws in America’s approach to many things nano. …

The article goes on to describe the GAO’s concern with “the missing middle” between basic research and laboratory scale prototypes on one hand, production at commercial scale on the other. This missing middle includes things like reliable manufacturing processes, and in the case of healthcare, clinical trials. The report also cites unfortunate policy lapses, like the lack of a grand nanomanufacturing strategy, failure to match competition from Russia and China, and failure to develop and adequately fund “nano-commons” where research, design, prototyping, and manufacturing can mutually profit from close proximity to each other.

Congressman Smith, unsurprisingly, believes there’s an Act for that — specifically the wordy but acronym-friendly Technology and Research Accelerating National Security and Future Economic Resiliency (TRANSFER) Act of 2013, which he co-sponsored. “The bill”, he believes, “will give researchers and universities incentives to partner with entrepreneurs and venture capitalists in order to move new technologies from the laboratory to the marketplace”.

The 125-page GAO report Nanomanufacturing: Emergence and Implications for U.S. Competitiveness, the Environment, and Human Health grew from a forum convened by the GAO in July 2013. A introductory paragraph states:

Although limited data on international investments made comparisons difficult, participants viewed the U.S. as likely leading in nanotechnology research and development (R&D) today. At the same time, they identified several challenges to U.S. competitiveness in nanomanufacturing, such as inadequate U.S. participation and leadership in international standard setting; the lack of a national vision for a U.S. nanomanufacturing capability; some competitor nations’ aggressive actions and potential investments; and funding or investment gaps in the United States … which may hamper U.S. innovators’ attempts to transition nanotechnology from R&D to full-scale manufacturing.

The report quite reasonably focuses on the nanomanufacturing gap that stands between advancements in current and near-term nanoscience and nanotechnology, on the one hand, and large-scale commercial developments, on the other. But in addition to near-term nanomanufacturing that will grow out of incremental advances in several areas of nanoscience, there is the component of advanced nanomanufacturing that has a longer development path, but will (IMHO) dominate the economy before mid-century. My quick search of the document did not turn up any mention of “molecular manufacturing”, “atomically precise manufacturing”, or “productive nanosystems”. A comprehensive view of the impact of nanotechnology on US competitiveness must (IMHO) embrace both incremental progress in nanomanufacturing for current and near-term applications, and advancement of nanomanufacturing ability to the ultimate limits of high throughput atomically precise manufacturing. For the history of why the U.S. lacks a national vision for advanced nanomanufacturing, check out chapter 13 “A funny thing happened on the way to the future …” of Eric Drexler’s 2013 book Radical Abundance [see also TEDx talk: “Transforming the Material Basis of Civilization”].
—James Lewis, PhD

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Bigger, stiffer, roomier molecular cages from structural DNA nanotechnology

The five cage-shaped DNA polyhedra here have struts stabilizing their legs, and this innovation allowed a Wyss Institute team to build by far the largest and sturdiest DNA cages yet. The largest, a hexagonal prism (right), is one-tenth the size of an average bacterium. Credit: Yonggang Ke/Harvard’s Wyss Institute

The use of structural DNA nanotechnology to build atomically precise scaffolds for positioning systems of molecular machines and other nanoscale functional elements [see, for example “Advancing nanotechnology by organizing functional components on addressable DNA scaffolds“] took a large step forward with the recent demonstration of the ability to build large, rigid three-dimensional DNA cages. The key innovation was the use of DNA origami to make struts to stabilize corners. A hat tip to ScienceDaily for reprinting this news release from Harvard University’s Wyss Institute “Roomy cages built from DNA“:

Move over, nanotechnologists, and make room for the biggest of the small. Scientists at the Harvard’s Wyss Institute have built a set of self-assembling DNA cages one-tenth as wide as a bacterium. The structures are some of the largest and most complex structures ever constructed solely from DNA, they report today’s online edition of Science [abstract].

Moreover, the scientists visualized them using a DNA-based super-resolution microscopy method — and obtained the first sharp 3D optical images of intact synthetic DNA nanostructures in solution.

In the future, scientists could potentially coat the DNA cages to enclose their contents, packaging drugs for delivery to tissues. And, like a roomy closet, the cage could be modified with chemical hooks that could be used to hang other components such as proteins or gold nanoparticles. This could help scientists build a variety of technologies, including tiny power plants, miniscule factories that produce specialty chemicals, or high-sensitivity photonic sensors that diagnose disease by detecting molecules produced by abnormal tissue.

“I see exciting possibilities for this technology,” said Peng Yin, Ph.D., a Core Faculty member at the Wyss Institute and Assistant Professor of Systems Biology at Harvard Medical School, and senior author of the paper.

Building with DNA

DNA is best known as a keeper of genetic information. But scientists in the emerging field of DNA nanotechnology are exploring ways to use it to build tiny structures for a variety of applications. These structures are programmable, in that scientists can specify the sequence of letters, or bases, in the DNA, and those sequences then determine the structure it creates.

So far most researchers in the field have used a method called DNA origami, in which short strands of DNA staple two or three separate segments of a much longer strand together, causing that strand to fold into a precise shape. DNA origami was pioneered in part by Wyss Institute Core Faculty member William Shih, Ph.D. [open access], who is also an Associate Professor in the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School and the Department of Cancer Biology at the Dana-Farber Cancer Institute.

Yin’s team has built different types of DNA structures, including a modular set of parts called single-stranded DNA tiles or DNA bricks [see “Arbitrarily complex 3D DNA nanostructures built from DNA bricks“]. Like LEGO® bricks, these parts can be added or removed independently. Unlike LEGO® bricks, they spontaneously self-assemble.

But for some applications, scientists might need to build much larger DNA structures than anyone has built so far. So, to add to their toolkit, Yin’s team sought much larger building blocks to match.

Engineering challenges

Yin and his colleagues first used DNA origami to create extra-large building blocks the shape of a photographer’s tripod. The plan was to engineer those tripod legs to attach end-to-end to form polyhedra — objects with many flat faces that are themselves triangles, rectangles, or other polygons.

But when Yin and the paper’s three lead authors, Ryosuke Iinuma, a former Wyss Institute Visiting Fellow, Yonggang Ke, Ph.D., a former Wyss Postdoctoral Fellow who is now an Assistant Professor of Biomedical Engineering at Georgia Institute of Technology and Emory University, and Ralf Jungman, Ph.D, a Wyss Postdoctoral Fellow, built bigger tripods and tried to assemble them into polyhedra, the large tripods’ legs would splay and wobble, which kept them from making polyhedra at all.

The researchers got around that problem by building in a horizontal strut to stabilize each pair of legs, just as a furniture maker would use a piece of wood to bridge legs of a wobbly chair.

To glue the tripod legs together end-to-end, they took advantage of the fact that matching DNA strands pair up and adhere to each other. They left a tag of DNA hanging off a tripod leg, and a matching tag on the leg of a different tripod that they wanted it to pair with.

The team programmed DNA to fold into sturdy tripods 60 times larger than previous DNA tripod-like building blocks and 400 times larger than DNA bricks. Those tripods then self-assembled into a specific type of three-dimensional polyhedron — all in a single test tube.

By adjusting the length of the strut, they built tripods that ranged from upright to splay-legged. More upright tripods formed polyhedra with fewer faces and sharper angles, such as a tetrahedron, which has four triangular faces. More splay-legged tripods formed polyhedra with more faces, such as a hexagonal prism, which is shaped like a wheel of cheese and has eight faces, including its top and bottom.

In all, they created five polyhedra: a tetrahedron, a triangular prism, a cube, a pentagonal prism, and a hexagonal prism.

Ultrasharp snapshots

After building the cages, the scientists visualized them using a DNA-based microscopy method Jungmann had helped developed called DNA-PAINT. In DNA-PAINT, short strands of modified DNA cause points on a structure to blink, and data from the blinking images reveal structures too small to be seen with a conventional light microscope. DNA-PAINT produced ultrasharp snapshots of the researchers’ DNA cages — the first 3D snapshots ever of single DNA structures in their native, watery environment.

“Bioengineers interested in advancing the field of nanotechnology need to devise manufacturing methods that build sturdy components in a highly robust manner, and develop self-assembly methods that enable formation of nanoscale devices with defined structures and functions,” said Wyss Institute Founding Director Don Ingber, M.D., Ph.D. “Peng’s DNA cages and his methods for visualizing the process in solution represent major advances along this path.”

As the above news release notes, “the cage could be modified with chemical hooks that could be used to hang other components such as proteins or gold nanoparticles.” With sides of 100 nm length, and a volume one one thousandth that of a typical bacterial cell, these ‘closets’ should be large enough to precisely assemble fairly complex assortments of nanoscale functional elements. Is it time to start thinking seriously about which assortments would be useful starting points for productive nanosystems?
—James Lewis, PhD

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Programmable nanoprocessors integrated into a nanowire nanocomputer

Credit: Yao et al. Proc Nat Acad Sci USA

Three years ago we noted “the world’s first programmable nanoprocessor” achieved by a collaboration between Harvard and MITRE [also, see further details here]. This year the same interdisciplinary team has taken further key steps toward a functioning nanoelectronic computer based on integrating several of the tiles that they first reported three years ago. A hat tip to KurzweilAI for reprinting this news release from MITRE “MITRE-Harvard Team’s Ultra-tiny Nanocomputer May Point the Way to Further Miniaturization in Industry“:

An interdisciplinary team of scientists and engineers from The MITRE Corporation and Harvard University has taken key steps toward ultra-small electronic computer systems that push beyond the imminent end of Moore’s Law, which states that the device density and overall processing power for computers will double every two to three years. In a paper … in the Proceedings of the National Academy of Sciences [abstract; full text PDF courtesy of the Lieber Research Group], the team describes how they designed and assembled, from the bottom up, a functioning, ultra-tiny control computer that is the densest nanoelectronic system ever built.

The ultra-small, ultra-low-power control processor—termed a nanoelectronic finite-state machine or “nanoFSM”—is smaller than a human nerve cell. It is composed of hundreds of nanowire transistors, each of which is a switch about ten-thousand times thinner than a human hair. The nanowire transistors use very little power because they are “nonvolatile.” That is, the switches remember whether they are on or off, even when no power is supplied to them.

In the nanoFSM, these nanoswitches are assembled and organized into circuits on several “tiles.” Together, the tiles route small electronic signals around the computer, enabling it to perform calculations and process signals that could be used to control tiny systems, such as miniscule medical therapeutic devices, other tiny sensors and actuators, or even insect-sized robots.

In 2011, the MITRE-Harvard team demonstrated a single such tiny tile capable of performing simple logic operations. In their recent collaboration they combined several tiles on a single chip to produce a first-of-its-kind complex, programmable nanocomputer.

“It was a challenge to develop a system architecture and nanocircuit designs that would pack the control functions we wanted into such a very tiny system,” according to Shamik Das, chief architect of the nanocomputer, who is also principal engineer and group leader of MITRE’s Nanosystems Group. “Once we had those designs, though, our Harvard collaborators did a brilliant job innovating to be able to realize them.”

Construction of this nanocomputer was made possible by significant advances in processes that assemble with extreme precision dense arrays of the many nanodevices required. These advances also made it possible to manufacture multiple copies of the nanoFSM, using a groundbreaking approach in which, for the first time, complex nanosystems can be economically assembled from the bottom up in close conformity to a preexisting design. Until now, this could be done using the industry’s expensive, top-down lithographic manufacturing methods, but not with bottom-up assembly.

For this reason, the nanoFSM and the means by which it was made represent a step toward extending the very economically important five-decade-long trend in miniaturization according to Moore’s Law, which has powered the electronics industry. Because of limitations on its conventional lithographic fabrication methods and on conventional transistors, many industry experts have suggested that the Moore’s Law trend soon may come to an end. Some assert that this might occur in as little as five years and have negative economic consequences, unless there are innovations in both device and fabrication technologies, such as those demonstrated by the nanoFSM.

James Ellenbogen, chief scientist for nanotechnology at MITRE and an expert in the development of computers integrated on the nanometer scale, said, “The nanoFSM and the new methods that were invented to build it are not the whole answer for the industry. However, I believe that they do incorporate important steps forward in two of the key areas the electronics industry has been focused upon in order to extend Moore’s Law.”

In addition to Das and Ellenbogen, the development team at MITRE included James Klemic, the corporation’s nanotechnology laboratory director. The researchers from MITRE—a pioneer in the nanotechnology field since 1992—collaborated with a three-person team at Harvard, led by Charles Lieber, a world-leading nanotechnology investigator.

In this work the three tiles integrate 180 programmable transistor nodes into both computing and memory elements, which were successfully reprogrammed to form a 2-bit full adder. It will be interesting to watch how far this promising approach can be scaled.
—James Lewis, PhD

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