DNA Nanomachines Are Opening Medicine to the World of Physics


Nano Machines 1 detectinghiv

When I imagine the inner workings of a robot, I think hard, cold mechanics running on physics: shafts, wheels, gears. Human bodies, in contrast, are more of a contained molecular soup operating on the principles of biochemistry.

Yet similar to robots, our cells are also attuned to mechanical forces—just at a much smaller scale. Tiny pushes and pulls, for example, can urge stem cells to continue dividing, or nudge them into maturity to replace broken tissues. Chemistry isn’t king when it comes to governing our bodies; physical forces are similarly powerful. The problem is how to tap into them.

In a new perspectives article in Science, Dr. Khalid Salaita and graduate student Aaron Blanchard from Emory University in Atlanta point to DNA as the solution. The team painted a futuristic picture of DNA mechanotechnology, in which we use DNA machines to control our biology. Rather than a toxic chemotherapy drip, for example, a cancer patient may one day be injected with DNA nanodevices that help their immune cells better grab onto—and snuff out—cancerous ones.

“For a long time,” said Salaita, “scientists have been good at making micro devices, hundreds of times smaller than the width of a human hair. It’s been more challenging to make functional nano devices, thousands of times smaller than that. But using DNA as the component parts is making it possible to build extremely elaborate nano devices because the DNA parts self-assemble.”

Just as the steam engine propelled civilization through the first industrial revolution, DNA devices may fundamentally change medicine, biological research, and the development of biomaterials, further merging man and machine.

Why DNA?

When picturing a tiny, whirling machine surveying the body, DNA probably isn’t the first candidate that comes to mind. Made up of long chains of four letters—A, T, C, and G—DNA is normally secluded inside a tiny porous “cage” in every cell, in the shape of long chains wrapped around a protein “core.”

Yet several properties make DNA a fascinating substrate for making mechano-machines, the authors said. One is its predictability: like soulmates, A always binds to T, and C with G. This chemical linking in turn forms the famous double helix structure. By giving the letters little chemical additions, or swapping them out altogether with unnatural synthetic letters, scientists have been able to form entirely new DNA assemblies, folded into various 3D structures.

Nano Machines 2 downloadRead More: Detecting HIV diagnostic antibodies with DNA nanomachines

Rather than an unbreakable, immutable chain, DNA components are more like Japanese origami paper, or Lego blocks. While they can’t make every single shape—try building a completely spherical Death Star out of Lego—the chemistry is flexible enough that scientists can tweak its structure, stiffness, and coiling by shifting around the letters or replacing them with entirely new ones.

 

The Rise of DNA Machines

In the late fall of 1980, Dr. Nadrian Seeman was relaxing at the campus pub at New York University when he noticed a mind-bending woodcut, Depth, by MC Escher. With a spark of insight, he realized that he could form similar lattice shapes using DNA, which would make it a lot easier for him to study the molecule’s shape. More than a decade later, his lab engineered the first artificial 3D nanostructure—a cube made out of DNA molecules. The field of DNA nanotechnology was born.

Originally considered a novelty, technologists rushed to make increasingly complex shapes, such as smiley faces, snowflakes, a tiny world map, and more recently, the world’s smallest playable tic-tac-toe set. It wasn’t just fun. Along the way, scientists uncovered sophisticated principles and engineering techniques to shape DNA strands into their desired structures, forming a blueprint of DNA engineering.

Then came the DNA revolution. Reading and writing the molecule from scratch became increasingly cheaper, making it easier to experiment with brand-new designs. Additional chemical or fluorescent tags or other modifications gave scientists a direct view of their creations. Rather than a fringe academic pursuit, DNA origami became accessible to most labs, and the number of devices rapidly exploded—devices that can sense, transmit, and generate mechanical forces inside cells.

“If you put together these three main components of mechanical devices, you begin to get hammers and cogs and wheels and you can start building nano machines,” said Salaita.

The Next Generation

Salaita is among several dozen labs demoing the practical uses of DNA devices.

For example, our cells are full of long-haul driver proteins that carry nutrients and other cargo throughout their interior by following specific highways (it eerily looks like a person walking down a tightrope). Just as too much traffic damages our roadways, changes in our cells’ logistical players can also harm the cell’s skeleton. Here, scientists have used DNA “handles” to measure force-induced changes like stretching, unfolding, and rupture of molecules involved in our cells’ distribution system to look for signs of trouble.

Then there are DNA tension sensors, which act like scales and other force gauges in our macroscopic world. Made up of a stretchable DNA “spring” to extend with force, and a fluorescent “ruler” that measures the extension, each sensor is anchored at one end (generally, the glass bottom of a Petri dish) and binds to a cell at the other. If the pulling force exceeds a certain threshold, the “spring” unfolds and quenches the fluorescent light in the ruler, giving scientists a warning that the cellular tugging is too strong.

The work may sound abstruse, but its implications are plenty. One is for CAR-T, the revolutionary cancer treatment that uses gene therapy to amp up immune cells with better “graspers” to target tumor cells. The “kiss of death” between graspers and tumors are extremely difficult to measure because it’s light and fleeting. Using a DNA tension sensor, the team was able to track the force during the interaction, which could help scientists engineer better CAR-T therapies. A similar construct, the DNA tension gauge tether, irreversibly ruptures under too much force. The gauge is used to track how stem cells develop into brain cells under mechanical forces, and how immune cells track down and recognize foreign invasion.

“[Immune] T cells are constantly sampling cells throughout your body using these mechanical tugs. They bind and pull on proteins on a cell’s surface and, if the bond is strong, that’s a signal that the T cell has found a foreign agent,” explained Salaita. DNA devices provide an unprecedented look at these forces in the immune system, which in turn could predict how strongly the body will mount an immune response.

To the authors, however, the most promising emerging DNA devices don’t just observe—they can also generate forces. DNA walkers, for example, uses DNA feet to transport (and sort) molecular cargo while walking down a track also made of DNA strands. When the feet “bind” to the “track” (A to T, C to G), it releases energy that propel the walker forward.

Even more exciting are self-assembling DNA machines. The field has DNA-based devices that “transmit, sense and generate mechanical forces,” the authors said. But eventually, their integration will produce nanomachines that “exert mechanical control over living systems.”

The Fourth Industrial Revolution

As costs keep dropping, the authors believe we’ll witness even more creative and sophisticated DNA nanomachines.

Several hiccups do stand in the way. Like other biomolecules, foreign DNA can be chopped up by the body’s immune system as an “invader.” However, the team believes that the limitation won’t be a problem in the next few years as biochemistry develops chemically-modified artificial DNA letters that resist the body’s scissors.

Another problem is that the DNA devices can generate very little force—less than a billionth the weight of a paperclip, which is a little too low to efficiently control forces in our cells. The authors have a solution here too: coupling many force-generating DNA units together, or engineer “translators” that can turn electrical energy into mechanical force—similar to the way our muscles work.

Fundamentally, any advancements in DNA mechanotechnology won’t just benefit medicine; they will also feed back into the design of nanomaterials. The “techniques, tools and design principles…are not specific” to DNA, the authors said. Add in computer-aided design templates, similar to those used in 3D printing, and “potentially anyone can dream up a nano-machine design and make it a reality,” said Salaita.

 

DNA ‘Origami’ takes Flight in Emerging Field of Nano Machines – “(a) … tool may eventually be used to fine tune immunotherapies for individual cancer patients”


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DNA mechanotechnology expands the opportunities for research involving biomedicine and materials sciences, says Khalid Salaita, right, professor of chemistry at Emory University and co-author of the article, along with Aaron Blanchard, left, a graduate student in the Salaita Lab. Credit: Emory University

Just as the steam engine set the stage for the Industrial Revolution, and micro transistors sparked the digital age, nanoscale devices made from DNA are opening up a new era in bio-medical research and materials science.

The journal Science describes the emerging uses of DNA  in a “Perspective” article by Khalid Salaita, a professor of chemistry at Emory University, and Aaron Blanchard, a graduate student in the Wallace H. Coulter Department of Biomedical Engineering, a joint program of Georgia Institute of Technology and Emory.

The article heralds a new field, which Blanchard dubbed “DNA mechanotechnology,” to engineer DNA machines that generate, transmit and sense  at the nanoscale.

“For a long time,” Salaita says, “scientists have been good at making micro devices, hundreds of times smaller than the width of a human hair. It’s been more challenging to make functional nano devices, thousands of times smaller than that. But using DNA as the component parts is making it possible to build extremely elaborate nano devices because the DNA parts self-assemble.”

DNA, or deoxyribonucleic acid, stores and transmits genetic information as a code made up of four chemical bases: adenine (A), guanine (G), cytosine (C) and thymine (T). The DNA bases have a natural affinity to pair up with each other—A with T and C with G. Synthetic strands of DNA can be combined with natural DNA strands from bacteriophages. By moving around the sequence of letters on the strands, researchers can get the DNA strands to bind together in ways that create different shapes. The stiffness of DNA strands can also easily be adjusted, so they remain straight as a piece of dry spaghetti or bend and coil like boiled spaghetti.

The idea of using DNA as a construction material goes back to the 1980s, when biochemist Nadrian Seeman pioneered DNA nanotechnology. This field uses strands DNA to make functional devices at the nanoscale. The ability to make these precise, three-dimensional structures began as a novelty, nicknamed DNA origami, resulting in objects such as a microscopic map of the world and, more recently, the tiniest-ever game of tic-tac-toe, played on a DNA board.

Work on novelty objects continues to provide new insights into the mechanical properties of DNA. These insights are driving the ability to make DNA machines that generate, transmit and sense mechanical forces.

“If you put together these three main components of mechanical devices, you begin to get hammers and cogs and wheels and you can start building nano machines,” Salaita says. “DNA mechanotechnology expands the opportunities for research involving biomedicine and materials science. It’s like discovering a new continent and opening up fresh territory to explore.”

Potential uses for such devices include drug delivery devices in the form of nano capsules that open up when they reach a target site, nano computers and nano robots working on nanoscale assembly lines.

The use of DNA self-assembly by the genomics industry, for biomedical research and diagnostics, is further propelling DNA mechanotechnology, making DNA synthesis inexpensive and readily available. “Potentially anyone can dream up a nano-machine design and make it a reality,” Salaita says.

He gives the example of creating a pair of nano scissors. “You know that you need two rigid rods and that they need to be linked by a pivot mechanism,” he says. “By tinkering with some open-source software, you can create this design and then go onto a computer and place an order to custom synthesize your design. You’ll receive your order in a tube. You simply put the tube contents into a solution, let your device self-assemble, and then use a microscope to see if it works the way you thought that it would.”

Salaita’s lab is one of only about 100 around the world working at the forefront of DNA mechanotechnology. He and Blanchard developed the world’s strongest synthetic DNA-based motor, which was recently reported in Nano Letters.

A key focus of Salaita’s research is mapping and measuring how cells push and pull to learn more about the mechanical forces involved in the human immune system.

Salaita developed the first DNA force gauges for cells, providing the first detailed view of the mechanical forces that one molecule applies to another molecule across the entire surface of a living cell. Mapping such forces may help to diagnose and treat diseases related to cellular mechanics. Cancer cells, for instance, move differently from normal cells, and it is unclear whether that difference is a cause or an effect of the disease.

In 2016, Salaita used these DNA force gauges to provide the first direct evidence for the mechanical forces of T cells, the security guards of the immune system. His lab showed how T cells use a kind of mechanical “handshake” or tug to test whether a cell they encounter is a friend or foe. These mechanical tugs are central to a T cell’s decision for whether to mount an immune response.

“Your blood contains millions of different types of T cells, and each T cell is evolved to detect a certain pathogen or foreign agent,” Salaita explains. “T cells are constantly sampling cells throughout your body using these mechanical tugs. They bind and pull on proteins on a cell’s surface and, if the bond is strong, that’s a signal that the T cell has found a foreign agent.”

Salaita’s lab built on this discovery in a paper recently published in the Proceedings of the National Academy of Sciences (PNAS). Work led by Emory chemistry graduate student Rong Ma refined the sensitivity of the DNA force gauges. Not only can they detect these mechanical tugs at a force so slight that it is nearly one-billionth the weight of a paperclip, they can also capture evidence of tugs as brief as the blink of an eye.

The research provides an unprecedented look at the mechanical forces involved in the immune system. “We showed that, in addition to being evolved to detect certain foreign agents, T cells will also apply very brief mechanical tugs to foreign agents that are a near match,” Salaita says. “The frequency and duration of the tug depends on how closely the foreign agent is matched to the T cell receptor.”

The result provides a tool to predict how strong of an immune response a T cell will mount. “We hope this tool may eventually be used to fine tune immunotherapies for individual cancer patients,” Salaita says. “It could potentially help engineer T  to go after particular .”


Computer scientists create reprogrammable molecular computing system – “Nano DNA Apps”


 

“Biology is proof that chemistry is inherently information-based and can store information that can direct algorithmic behavior at the molecular level,” he says.

Computer scientists at Caltech have designed DNA molecules that can carry out reprogrammable computations, for the first time creating so-called algorithmic self-assembly in which the same “hardware” can be configured to run different “software.”

In a paper published in Nature on March 21, a team headed by Caltech’s Erik Winfree (PhD ’98), professor of computer science, computation and neural systems, and bioengineering, showed how the DNA computations could execute six-bit algorithms that perform simple tasks. The system is analogous to a computer, but instead of using transistors and diodes, it uses molecules to represent a six-bit binary number (for example, 011001) as input, during computation, and as output. One such algorithm determines whether the number of 1-bits in the input is odd or even, (the example above would be odd, since it has three 1-bits); while another determines whether the input is a palindrome; and yet another generates random numbers.

 

“Think of them as nano apps,” says Damien Woods, professor of computer science at Maynooth University near Dublin, Ireland, and one of two lead authors of the study. “The ability to run any type of software program without having to change the hardware is what allowed computers to become so useful. We are implementing that idea in molecules, essentially embedding an algorithm within chemistry to control chemical processes.”

The system works by self-assembly: small, specially designed DNA strands stick together to build a logic circuit while simultaneously executing the circuit algorithm. Starting with the original six bits that represent the input, the system adds row after row of molecules–progressively running the algorithm.

 

Modern digital electronic computers use electricity flowing through circuits to manipulate information; here, the rows of DNA strands sticking together perform the computation. The end result is a test tube filled with billions of completed algorithms, each one resembling a knitted scarf of DNA, representing a readout of the computation. The pattern on each “scarf” gives you the solution to the algorithm that you were running. The system can be reprogrammed to run a different algorithm by simply selecting a different subset of strands from the roughly 700 that constitute the system.

 

“We were surprised by the versatility of programs we were able to design, despite being limited to six-bit inputs,” says David Doty, fellow lead author and assistant professor of computer science at the University of California, Davis.

“When we began experiments, we had only designed three programs. But once we started using the system, we realized just how much potential it has. It was the same excitement we felt the first time we programmed a computer, and we became intensely curious about what else these strands could do. By the end, we had designed and run a total of 21 circuits.”

 

The researchers were able to experimentally demonstrate six-bit molecular algorithms for a diverse set of tasks. In mathematics, their circuits tested inputs to assess if they were multiples of three, performed equality checks, and counted to 63. Other circuits drew “pictures” on the DNA “scarves,” such as a zigzag, a double helix, and irregularly spaced diamonds.

Probabilistic behaviors were also demonstrated, including random walks, as well as a clever algorithm (originally developed by computer pioneer John von Neumann) for obtaining a fair 50/50 random choice from a biased coin.

 

Both Woods and Doty were theoretical computer scientists when beginning this research, so they had to learn a new set of “wet lab” skills that are typically more in the wheelhouse of bioengineers and biophysicists.

“When engineering requires crossing disciplines, there is a significant barrier to entry,” says Winfree. “Computer engineering overcame this barrier by designing machines that are reprogrammable at a high level–so today’s programmers don’t need to know transistor physics. Our goal in this work was to show that molecular systems similarly can be programmed at a high level, so that in the future, tomorrow’s molecular programmers can unleash their creativity without having to master multiple disciplines.”

“Unlike previous experiments on molecules specially designed to execute a single computation, reprogramming our system to solve these different problems was as simple as choosing different test tubes to mix together,” Woods says. “We were programming at the lab bench.”

Although DNA computers have the potential to perform more complex computations than the ones featured in the Nature paper, Winfree cautions that one should not expect them to start replacing the standard silicon microchip computers. That is not the point of this research.

“These are rudimentary computations, but they have the power to teach us more about how simple molecular processes like self-assembly can encode information and carry out algorithms. Biology is proof that chemistry is inherently information-based and can store information that can direct algorithmic behavior at the molecular level,” he says.

 

Diverse and robust molecular algorithms using reprogrammable DNA self-assembly

Damien Woods, David Doty, Cameron Myhrvold, Joy Hui, Felix Zhou, Peng Yin & Erik Winfree
Naturevolume 567, pages366–372 (2019)

 

NEW NANOTECH DRIVES HEALING BY “TALKING” TO WOUNDS


A Time To Heal

Researchers from Imperial College London have created a new molecule that can “talk” to the cells in the area near injured tissues to encourage wound healing.

“This intelligent healing is useful during every phase of the healing process, has the potential to increase the body’s chance to recover, and has far-reaching uses on many different types of wounds,” lead researcher Ben Almquist said in a news release.

Setting A TrAP

The Imperial team describes the wound-healing molecules, which it calls traction force-activated payloads (TrAPs), in a study published Monday in the journal Advanced Materials.

The first step to creating TrAPs was folding segments of DNA into aptamers, which are three-dimensional shapes that latch tightly to proteins. The researchers then added a “handle” to one end of the aptamer.

As cells navigated the area near a wound during lab testing, they would pull on this handle, causing the aptamer to open and release proteins that encouraged wound healing. By changing the handle, the researchers found they could control which cells activated the TrAPs.

According to Almquist, “TrAPs provide a flexible method of actively communicating with wounds, as well as key instructions when and where they are needed.”

To The Clinic

It can take a long time for research to move from the laboratory to the clinical trial stage, but the TrAPs team might be able to speed along the path. That’s because aptamers are already used for drug delivery, meaning they’re already considered safe for human use.

TrAPs are also fairly straightforward to create, meaning it wouldn’t be difficult to scale the technology to industrial levels. According to the researchers’ paper, doctors could then deliver the TrAPs via anything from collagen sponges to polyacrylamide gels. So if future testing goes well, the molecules could soon change how we heal a variety of wounds.

READ MORE: New Material Could ‘Drive Wound Healing’ Using the Body’s Inbuilt Healing System [Imperial College London]

More on aptamers: New Nanobots Kill Cancerous Tumors by Cutting off Their Blood Supply

Story Source:

Materials provided by Imperial College London. Original written by Caroline Brogan. Note: Content may be edited for style and length.


Journal Reference:

  1. Anna Stejskalová, Nuria Oliva, Frances J. England, Benjamin D. Almquist. Biologically Inspired, Cell‐Selective Release of Aptamer‐Trapped Growth Factors by Traction Forces. Advanced Materials, 2018 DOI: 10.1002/adma.201806380

Synthetic organisms are about to challenge what ’being’ and ‘alive’ really means


We need to begin a serious debate about whether artificially evolved humans are our future, and if we should put an end to these experiments before it is too late.

In 2016, Craig Venter and his team at Synthetic Genomics announced that they had created a lifeform called JCVI-syn3.0, whose genome consisted of only 473 genes.

This stripped-down organism was a significant breakthrough in the development of artificial life as it enabled us to understand more fully what individual genes do. (In the case of JCVI-syn3.0, most of them were used to create RNA and proteins, preserve genetic fidelity during reproduction and create the cell membrane.

The functions of about a third remain a mystery.)

Venter’s achievement followed an earlier breakthrough in 2014, when Floyd Romesberg at Romesberg Lab in California succeeded in creating xeno nucleic acid (XNA), a synthetic alternative to DNA, using amino acids not found among the naturally occurring four nucleotides: adenine, cytosine, guanine and thymine. 

And, most recently we have seen huge advances in the use of CRISPR, a gene-editing tool that allows substitution or injection of DNA sequences at chosen locations in a genome.

Read More: Why Bill Gates is Betting on this Synthetic Biology Start-Up

Together, these developments mean that in 2019 we will have to take seriously the possibility of our developing multicellular artificial life, and we will need to start thinking about the ethical and philosophical challenges such a possibility brings up.

In the near future we can reasonably anticipate that a large number of unnatural single-cell life forms will be created using artificially edited genomes to correct for genetic defects or to add new features to an organism’s phenotype.

It is already possible to design bacterial forms, for example, that can metabolise pollutants or produce particular substances.

We can also anticipate that new life forms may be created that have never existed in nature through the use of conventional and perhaps artificially arranged codons (nucleotide sequences that manage protein synthesis).

These are likely to make use of the conventional machinery of mitotic cell reproduction and of conventional ribosomes, creating proteins through RNA or XNA interpretation.

And there will be increasing pressures to continue this research. We may need to accelerate the evolution of terrestrial life forms, for example, including homo sapiens, so that they carry traits and capabilities needed for life in space or even on our own changing planet. 

All of this will bring up serious issues as to how we see ourselves – and behave – as a species.

While the creation of multicellular organisms that are capable of sexual reproduction is still a long way off, in 2019 we will need to begin a serious debate about whether artificially evolved humans are our future, and if we should put an end to these experiments before it is too late.

 Vint Cerf of ‘Wired’

MIT Team invents method to shrink objects to the nanoscale – “Implosion Manufacturing” – Applications from Optics to Medicine to Robotics – Materials from Quantum Dots, Metals and DNA


MIT Implosion mfg mitteaminvenAccording to professor Ed Boyden, many research labs are already stocked with the equipment required for this kind of fabrication. Credit: The researchers

” … These tiny structures could have applications in many fields, from optics to medicine to robotics, the researchers say.”

MIT researchers have invented a way to fabricate nanoscale 3-D objects of nearly any shape. They can also pattern the objects with a variety of useful materials, including metals, quantum dots, and DNA.

“It’s a way of putting nearly any kind of material into a 3-D pattern with nanoscale precision,” says Edward Boyden, an associate professor of biological engineering and of brain and cognitive sciences at MIT.

Using the , the researchers can create any shape and structure they want by patterning a  with a laser. After attaching other useful materials to the scaffold, they shrink it, generating structures one thousandth the volume of the original.

These tiny structures could have applications in many fields, from optics to medicine to robotics, the researchers say. The technique uses equipment that many biology and materials science labs already have, making it widely accessible for researchers who want to try it.

Boyden, who is also a member of MIT’s Media Lab, McGovern Institute for Brain Research, and Koch Institute for Integrative Cancer Research, is one of the senior authors of the paper, which appears in the Dec. 13 issue of Science. The other senior author is Adam Marblestone, a Media Lab research affiliate, and the paper’s lead authors are graduate students Daniel Oran and Samuel Rodriques.

MIT-Implosion-Fabrication-01Implosion fabrication

Existing techniques for creating nanostructures are limited in what they can accomplish. Etching patterns onto a surface with light can produce 2-D nanostructures but doesn’t work for 3-D structures. It is possible to make 3-D nanostructures by gradually adding layers on top of each other, but this process is slow and challenging. And, while methods exist that can directly 3-D print nanoscale objects, they are restricted to specialized materials like polymers and plastics, which lack the functional properties necessary for many applications. Furthermore, they can only generate self-supporting structures. (The technique can yield a solid pyramid, for example, but not a linked chain or a hollow sphere.)

To overcome these limitations, Boyden and his students decided to adapt a technique that his lab developed a few years ago for high-resolution imaging of brain tissue. This technique, known as expansion microscopy, involves embedding tissue into a hydrogel and then expanding it, allowing for high resolution imaging with a regular microscope. Hundreds of research groups in biology and medicine are now using expansion microscopy, since it enables 3-D visualization of cells and tissues with ordinary hardware.

By reversing this process, the researchers found that they could create large-scale objects embedded in expanded hydrogels and then shrink them to the nanoscale, an approach that they call “implosion fabrication.”

As they did for , the researchers used a very absorbent material made of polyacrylate, commonly found in diapers, as the scaffold for their nanofabrication process. The scaffold is bathed in a solution that contains molecules of fluorescein, which attach to the scaffold when they are activated by laser light.

Using two-photon microscopy, which allows for precise targeting of points deep within a structure, the researchers attach fluorescein molecules to specific locations within the gel. The fluorescein molecules act as anchors that can bind to other types of molecules that the researchers add.

“You attach the anchors where you want with light, and later you can attach whatever you want to the anchors,” Boyden says. “It could be a quantum dot, it could be a piece of DNA, it could be a gold nanoparticle.”

“It’s a bit like film photography—a latent image is formed by exposing a sensitive material in a gel to light. Then, you can develop that latent image into a real image by attaching another material, silver, afterwards. In this way implosion fabrication can create all sorts of structures, including gradients, unconnected structures, and multi-material patterns,” Oran says.

Once the desired molecules are attached in the right locations, the researchers shrink the entire structure by adding an acid. The acid blocks the negative charges in the polyacrylate gel so that they no longer repel each other, causing the gel to contract. Using this technique, the researchers can shrink the objects 10-fold in each dimension (for an overall 1,000-fold reduction in volume). This ability to shrink not only allows for increased resolution, but also makes it possible to assemble materials in a low-density scaffold. This enables easy access for modification, and later the material becomes a dense solid when it is shrunk.

“People have been trying to invent better equipment to make smaller nanomaterials for years, but we realized that if you just use existing systems and embed your  in this gel, you can shrink them down to the nanoscale, without distorting the patterns,” Rodriques says.

Currently, the researchers can create objects that are around 1 cubic millimeter, patterned with a resolution of 50 nanometers. There is a tradeoff between size and resolution: If the researchers want to make larger objects, about 1 cubic centimeter, they can achieve a resolution of about 500 nanometers. However, that resolution could be improved with further refinement of the process, the researchers say.

Better optics

The MIT team is now exploring potential applications for this technology, and they anticipate that some of the earliest applications might be in optics—for example, making specialized lenses that could be used to study the fundamental properties of light. This technique might also allow for the fabrication of smaller, better lenses for applications such as cell phone cameras, microscopes, or endoscopes, the researchers say. Farther in the future, the researchers say that this approach could be used to build nanoscale electronics or robots.

“There are all kinds of things you can do with this,” Boyden says. “Democratizing nanofabrication could open up frontiers we can’t yet imagine.”

Many research labs are already stocked with the equipment required for this kind of fabrication. “With a laser you can already find in many biology labs, you can scan a pattern, then deposit metals, semiconductors, or DNA, and then shrink it down,” Boyden says.

 Explore further: High-resolution imaging with conventional microscopes

More information: D. Oran el al., “3D nanofabrication by volumetric deposition and controlled shrinkage of patterned scaffolds,” Science (2018). science.sciencemag.org/cgi/doi … 1126/science.aau5119

T.E. Long el al., “Printing nanomaterials in shrinking gels,” Science (2018). science.sciencemag.org/cgi/doi … 1126/science.aav5712

 

‘Swiss army knife’ Nanovaccine carries multiple weapons to battle tumors – cancer


Swiss Army Knife of Nano Ps 171129163851_1_540x360
Source: National Institute of Biomedical Imaging and Bioengineering
Summary: Researchers have developed a synergistic cancer nanovaccine packing DNA and RNA sequences that modulate the immune response, along with anti-tumor antigens, into one small nanoparticle.
(Above) Large particles (left) containing the DNA and RNA components are coated with electronically charged molecules that shrink the particle. The tumor-specific neoantigen is then complexed with the surface to complete construction of the nanovaccine. Upper left: electron micrograph of large particle. Credit: Zhu, et al. Nat Comm.

 

 

Scientists are using their increasing knowledge of the complex interaction between cancer and the immune system to engineer increasingly potent anti-cancer vaccines. The nanovaccine produced an immune response that specifically killed tumor tissue, while simultaneously inhibiting tumor-induced immune suppression to block lung tumor growth in a mouse model of metastatic colon cancer.

Now researchers at the National Institute of Biomedical Imaging and Bioengineering (NIBIB) have developed a synergistic nanovaccine packing DNA and RNA sequences that modulate the immune response, along with anti-tumor antigens, into one small nanoparticle. The nanovaccine produced an immune response that specifically killed tumor tissue, while simultaneously inhibiting tumor-induced immune suppression. Together this blocked lung tumor growth in a mouse model of metastatic colon cancer.

The molecular dance between cancer and the immune system is a complex one and scientists continue to identify the specific molecular pathways that rev up or tamp down the immune system. Biomedical engineers are using this knowledge to create nanoparticles that can carry different molecular agents that target these pathways. The goal is to simultaneously stimulate the immune system to specifically attack the tumor while also inhibiting the suppression of the immune system, which often occurs in cancer patients. The aim is to press on the gas pedal of the immune system while also releasing the emergency brake.

A key hurdle is to design a system to reproducibly and efficiently create a nanoparticle loaded with multiple agents that synergize to mount an enhanced immune attack on the tumor. Engineers at the NIBIB report the development and testing of such a nanovaccine in the November issue of Nature Communications.

Making all the parts fit

Guizhi Zhu, Ph.D., a post-doctoral fellow in the NIBIB Laboratory of Molecular Imaging and Nanomedicine (LOMIN) and lead author on the study, explains the challenge. “We are very excited about putting multiple cooperating molecules that have anti-cancer activity into one nanovaccine to increase effectiveness. However, the bioengineering challenge is fitting everything in to a small particle and designing a way to maintain its structural integrity and biological activity.”

Zhu and his colleagues have created what they call a “self-assembling, intertwining DNA-RNA nanocapsule loaded with tumor neoantigens.” They describe it as a synergistic vaccine because the components work together to stimulate and enhance an immune attack against a tumor.

The DNA component of the vaccine is known to stimulate immune cells to work with partner immune cells for antitumor activation. The tumor neoantigens are pieces of proteins that are only present in the tumor; so, when the DNA attracts the immune cells, the immune cells interact with the tumor neoantigens and mount an expanded and specific immune response against the tumor. The RNA is the component that inhibits suppression of the immune system. The engineered RNA binds to and degrades the tumor’s mRNA that makes a protein called STAT3. Thus, the bound mRNA is blocked from making STAT3, which may suppress the immune system. The result is an enhanced immune response that is specific to the tumor and does not harm healthy tissues.

In addition to engineering a system where the DNA, RNA and tumor neoantigens self-assemble into a stable nanoparticle, an important final step in the process is shrinking the particle. Zhu explains: “Shrinking the particle is a critical step for activating an immune response. This is because a very small nanoparticle can more readily move through the lymphatic vessels to reach the parts of the immune system such as lymph nodes. A process that is essential for immune activation.”

The method for shrinking also had to be engineered. This was achieved by coating the particle with a positively charged polypeptide that interacts with the negatively charged DNA and RNA components to condense it to one-tenth of its original size.

Testing the nanovaccine

To create a model of metastatic colon cancer, the researchers injected human colon cancer cells into the circulation of mice. The cells infiltrate different organs and grow as metastatic colon cancer. One of the prime sites of metastasis is the lung.

The nanovaccine was injected under the skin of the mice 10, 16, and 22 days after the colon cancer cells were injected. To compare to the nanovaccine, two control groups of mice were analyzed; one group was injected with just the DNA and the neoantigen in solution but not formed into a nanovaccine particle, and the second control group was injected with an inert buffer solution.

At 40 days into the experiment, lung tumors from the nanovaccine-treated and the control groups were assessed by PET-CT imaging, and then removed and weighed. In mice treated with the nanovaccine, tumors were consistently one tenth the size of the tumors that were found in mice in both control groups.

Further testing revealed that mice receiving the nanovaccine had a significant increase in circulating cytotoxic T lymphocytes (CTLs) that specifically targeted the neoantigen on the colon cancer cells. CTLs are cells that attack and kill virus-infected cells and those damaged in other ways, such as cancerous cells.

An important aspect of the nanovaccine approach is that it mounts an anti-tumor immune response that circulates through the system, and therefore is particularly valuable for finding and inhibiting metastatic tumors growing throughout the body.

The researchers view their nanovaccine as an important part of eventual therapies combining immunotherapy with other cancer killing approaches.

Story Source:

Materials provided by National Institute of Biomedical Imaging and BioengineeringNote: Content may be edited for style and length.


Journal Reference:

  1. Guizhi Zhu, Lei Mei, Harshad D. Vishwasrao, Orit Jacobson, Zhantong Wang, Yijing Liu, Bryant C. Yung, Xiao Fu, Albert Jin, Gang Niu, Qin Wang, Fuwu Zhang, Hari Shroff, Xiaoyuan Chen. Intertwining DNA-RNA nanocapsules loaded with tumor neoantigens as synergistic nanovaccines for cancer immunotherapyNature Communications, 2017; 8 (1) DOI: 10.1038/s41467-017-01386-7

DNA and Nanotechnology: Protein ‘Rebar’ could help make Error-Free Nanostructures: NIST


DNA proteinrebarThe protein RecA (purple units), wraps around and fortifies double-stranded DNA, enabling scientists to build large structures with the genetic material. Credit: NIST

DNA is the stuff of life, but it is also the stuff of nanotechnology. Because molecules of DNA with complementary chemical structures recognize and bind to one another, strands of DNA can fit together like Lego blocks to make nanoscale objects of complex shape and structure.

But researchers need to work with much larger assemblages of DNA to realize a key goal: building durable miniature devices such as biosensors and drug-delivery containers. That’s been difficult because long chains of DNA are floppy and the standard method of assembling long chains is prone to error.

Using a DNA-binding protein called RecA as a kind of nanoscale rebar, or reinforcing bar, to support the floppy DNA scaffolding, researchers at the National Institute of Standards and Technology (NIST) have constructed several of the largest rectangular, linear and other shapes ever assembled from DNA. The structures can be two to three times larger than those built using standard DNA self-assembly techniques.

In addition, because the new method requires fewer chemically distinct pieces to build organized structures than the standard technique, known as DNA origami, it is likely to reduce the number of errors in constructing the shapes. That’s a big plus for the effort to produce reliable DNA-based devices in large quantities, said NIST researcher Alex Liddle.

Although RecA’s ability to bind to double-stranded DNA has been known for years, the NIST team is the first to integrate filaments of this protein into the assembly of DNA structures. The addition of RecA offers a particular advantage: Once one unit of the protein binds to a small segment of double-stranded DNA, it automatically attracts other units to line up alongside it, in the same way that bar magnets will join end-to-end. Like bricks filling out a foundation, RecA lines the entire length of the DNA strand, stretching, widening and strengthening it. A floppy, 2-nanometer-wide strand of DNA can transform into a rigid structure more than four times as wide.

“The RecA method greatly extends the ability of DNA self-assembly methods to build larger and more sophisticated structures,” said NIST’s Daniel Schiffels.

Schiffels, Liddle and their colleague Veronika Szalai describe their work in a recent article in ACS Nano.

The new method incorporates the DNA origami technique and goes beyond it, according to Liddle. In DNA origami, short strands of DNA that have a specific sequence of four base pairs are used as staples to tie together long sections of DNA. To make the skinny DNA skeleton stronger and thicker, the strand may loop back on itself, quickly using up the long string.

If DNA origami is all about the folding, Liddle likened his team’s new method to building a room, starting with a floor plan. The location of the short, single-stranded pieces of DNA that act as staples mark the corners of the room. Between the corners lies a long, skinny piece of single-stranded DNA. The enzyme DNA polymerase transforms a section of the long piece of single-stranded DNA into the double-stranded version of the molecule, a necessary step because RecA only binds strongly to double-stranded DNA. Then RecA assembles all along the double strand, reinforcing the DNA  and limiting the need for extra staples to maintain its shape.

With fewer staples required, the RecA  is likely able to build organized structures with fewer errors than DNA origami, Liddle said.

 Explore further: The promise of nanomanufacturing using DNA origami

More information: Daniel Schiffels et al. Molecular Precision at Micrometer Length Scales: Hierarchical Assembly of DNA–Protein Nanostructures, ACS Nano (2017). DOI: 10.1021/acsnano.7b00320

 

Arizona State University ~ ‘Living Computers’ from RNA for Nanotechnology


RNA Nano 2 bd1d43755f5067d16cb5985bd7de8ea1a3a38212

Researchers from Arizona State University have demonstrated that living cells can be induced to carry out complex computations in the manner of tiny robots or computers.

It’s an example of engineers and biologists coming together to create an innovative solution to the performing of calculations. The implications are a potential game-changer for intelligent drug design and smart drug delivery. Other fields that could be affected include green energy production, low-cost diagnostic technologies and the development of futuristic nanomachines to be used in gene-editing. ASU xximage_1.png.pagespeed.ic.dPihifYIDEThe basis of the new technology is the natural interactions between nucleic acid; in this case the predictable and programmable RNA-RNA interactions. RNA is ribonucleic acid, an important molecule with long chains of nucleotides.
A nucleotide contains a nitrogenous base, a ribose sugar, and a phosphate. RNA is involved with the coding, decoding, regulation, and expression of genes. This builds on earlier work where DNA and RNA, the molecules of life, where demonstrated as being able to perform computer-like computations by Leonard Adleman (University of Southern California) in 1994 (“Molecular Computation of Solutions To Combinatorial Problems.”)
Atomic structure of the 50S Large Subunit of the Ribosome. Proteins are colored in blue and RNA in o...

Atomic structure of the 50S Large Subunit of the Ribosome. Proteins are colored in blue and RNA in orange. RNA is central to the synthesis of proteins. Wikipedia / Vossman
From this basis, lead researcher Professor Alex Green has used computer software to design RNA sequences that behave the way researchers want them to in a cell. This makes the design process a much faster.RNA Nano 3 RNAThe output is circuit designs, which look like conventional electronic circuits, but which self-assemble inside bacterial cells. This allows the cells to sense incoming messages and respond to them by producing a computational output. To test this out, the researchers worked with specialized circuits called logic gates. The tiny circuit switches were tripped when messages (RNA fragments) which attached themselves to their complementary RNA sequences in the cellular circuit. This activated the logic gate and produced an output. A series of more complex logic gates were then designed, to respond to multiple inputs. Here logic gates known as AND, OR and NOT were designed.
The video below explains more about these switches:

From this the scientists developed the first ribocomputing devices capable of four-input AND, six-input OR and a 12-input device able to carry out a complex combination of AND, OR and NOT logic known as disjunctive normal form expression.The great strength of the new method is with its ability to perform many operations at the same time. This capacity for parallel processing allows for faster and more sophisticated computation.The example, of meshing engineering and biology together, is part of an emerging field called synthetic biology, and it is one of the fastest growing areas of scientific research. In a sense, synthetic biology is a biology-based “toolkit”. According to the European research group ERBC the science deploys abstraction, standardization, and automated construction to change how we build biological systems and expand the range of possible products. One such example of what a highly accurate platform like this could do is with diagnosing viruses the Zika virus.The research has been published in the journalNature under the title “Complex cellular logic computation using ribocomputing devices.”

 

Laser activated gold pyramids could deliver drugs, DNA into cells without harm


Harvard DNA Delivery 170323150417_1_540x360

Summary: The ability to deliver cargo like drugs or DNA into cells is essential for biological research and disease therapy but cell membranes are very good at defending their territory. Researchers have developed various methods to trick or force open the cell membrane but these methods are limited in the type of cargo they can deliver and aren’t particularly efficient. Harvard School of Engineering and Applied Sciences 

The ability to deliver cargo like drugs or DNA into cells is essential for biological research and disease therapy but cell membranes are very good at defending their territory. Researchers have developed various methods to trick or force open the cell membrane but these methods are limited in the type of cargo they can deliver and aren’t particularly efficient.

Now, researchers from the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS) have developed a new method using gold microstructures to deliver a variety of molecules into cells with high efficiency and no lasting damage. The research is published in ACS Nano.

“Being able to effectively deliver large and diverse cargos directly into cells will transform biomedical research,” said Nabiha Saklayen, a PhD candidate in the Mazur Lab at SEAS and first author of the paper. “However, no current single delivery system can do all the things you need to do at once. Intracellular delivery systems need to be highly efficient, scalable, and cost effective while at the same time able to carry diverse cargo and deliver it to specific cells on a surface without damage. It’s a really big challenge.”

In previous research, Saklayen and her collaborators demonstrated that gold, pyramid-shaped microstructures are very good at focusing laser energy into electromagnetic hotspots. In this research, the team used a fabrication method called template stripping to make surfaces — about the size of a quarter — with 10 million of these tiny pyramids.

“The beautiful thing about this fabrication process is how simple it is,” said Marinna Madrid, coauthor of the paper and PhD candidate in the Mazur Lab. “Template-stripping allows you to reuse silicon templates indefinitely. It takes less than a minute to make each substrate, and each substrate comes out perfectly uniform. That doesn’t happen very often in nanofabrication.”

Harvard DNA Delivery 170323150417_1_540x360
A scanning-electron microscope image of chemically-fixed HeLa cancer cells on the substrate. The tips of the pyramids create tiny holes in the cell membranes, allowing molecular cargo to diffuse into the cells. Credit: Harvard SEAS

The team cultured HeLa cancer cells directly on top of the pyramids and surrounded the cells with a solution containing molecular cargo.

Using nanosecond laser pulses, the team heated the pyramids until the hotspots at the tips reached a temperature of about 300 degrees Celsius. This very localized heating — which did not affect the cells — caused bubbles to form right at the tip of each pyramid. These bubbles gently pushed their way into the cell membrane, opening brief pores in the cell and allowing the surrounding molecules to diffuse into the cell.

“We found that if we made these pores very quickly, the cells would heal themselves and we could keep them alive, healthy and dividing for many days,” Saklayen said.

Each HeLa cancer cell sat atop about 50 pyramids, meaning the researchers could make about 50 tiny pores in each cell. The team could control the size of the bubbles by controlling the laser parameters and could control which side of the cell to penetrate.

The molecules delivered into the cell were about the same size as clinically relevant cargos, including proteins and antibodies.

Next, the team plans on testing the methods on different cell types, including blood cells, stem cells and T cells. Clinically, this method could be used in ex vivo therapies, where unhealthy cells are taken out of the body, given cargo like drugs or DNA, and reintroduced into the body.

“This work is really exciting because there are so many different parameters we could optimize to allow this method to work across many different cell types and cargos,” said Saklayen. “It’s a very versatile platform.”

Harvard’s Office of Technology Development has filed patent applications and is considering commercialization opportunities.

“It’s great to see how the tools of physics can greatly advance other fields, especially when it may enable new therapies for previously difficult to treat diseases,” said Eric Mazur, the Balkanski Professor of Physics and Applied Physics and senior author of the paper.

This research was supported by the National Science Foundation and the Howard Hughes Medical Institute. It was coauthored by Marinus Huber, Marinna Madrid, Valeria Nuzzo, Daryl Inna Vulis, Weilu Shen, Jeffery Nelson, Arthur McClelland and Alexander Heisterkamp.


Story Source:

Materials provided by Harvard School of Engineering and Applied Sciences. Note: Content may be edited for style and length.


Journal Reference:

  1. Nabiha Saklayen, Marinus Huber, Marinna Madrid, Valeria Nuzzo, Daryl I. Vulis, Weilu Shen, Jeffery Nelson, Arthur A. McClelland, Alexander Heisterkamp, Eric Mazur. Intracellular Delivery Using Nanosecond-Laser Excitation of Large-Area Plasmonic Substrates. ACS Nano, 2017; DOI: 10.1021/acsnano.6b08162