Stanford Bioengineers Create Remote-Controlled Nanoscale Protein Motors

Stanford bioengineers create remote-controlled nanoscale protein motors

A team led by Assistant Professor Zev Bryant builds molecular motors to further the study of cell function.

In every cell in your body, tiny protein motors are toiling away to keep you going. Moving muscles, dividing cells, twisting DNA – they are the workhorses of biology. But there is still uncertainty about how they function. To help biologists in the quest to know more, a team of Stanford bioengineers has designed a suite of protein motors that can be controlled remotely by light.

"Biology is full of these nanoscale machines that can perform complex tasks," said Zev Bryant, an assistant professor of bioengineering and leader of the team. "We want to understand how they can convert chemical energy into mechanical work and perform their specific tasks in cells."

Bryant's team, including doctoral student Muneaki Nakamura, designed blueprints for protein motors that would respond to light. Splicing together DNA from different organisms such as pig, slime mold and oat – the oat had the light-detecting module – the bioengineers created DNA codes for each of their protein motors.

Assistant Professor Zev Bryant and his team are creating tiny protein motors that can be controlled remotely by light. (Linda A. Cicero / Stanford News Service)

The remote-controlled nanomotors are described by Nakamura, Bryant and their colleagues in a paper that appeared online Aug. 3 in Nature Nanotechnology.

When exposed to light, the new protein motors change direction or speed. "It's pretty fine spatial control; you can decide where the light is and where it isn't and control motors in this very exquisite way," Bryant said. Being able to control the motors in real time should be a boon for cell and developmental biologists trying to study forces and motion in living things.

"It's an entirely new project for us to be moving inside cells and organisms, and to be working more closely with biologists," Bryant said. Now that he and his team have a basic blueprint, they will be able to customize these motors for biologists who are looking into specific tasks.

"In a future phase of our research, I hope that we can provide cell biologists with tools that allow them to change very specifically the properties of molecular motors in their cellular contexts," Bryant said.

Protein filaments (labeled in green and red) glide on the surface of a microscope coverslip, their motion driven by engineered molecular motors. The motors shift gears when illuminated with blue light, causing the motion to slow down. (Video: Bryant Lab)

There's also the possibility of using the controllable motors outside of biology, in diagnostic devices, for example. Bryant noted that researchers have worked on harnessing molecular motors to perform functions similar to their biological roles, "transporting molecules, sorting molecules, and concentrating molecules."

But first and foremost, Bryant is rebuilding these motors – incorporating features never before seen in biology – in an effort to illuminate their true nature.

"Evolution takes a basic design and makes motors that are fast and motors that are slow and motors that move long distances," Bryant said. "We've tried to build diverse motors and really challenge our understanding by pushing ourselves outside of what's already been done by evolution."

Shara Tonn is an intern at the Stanford News Service.

Tuesday, August 5, 2014

ORIGINAL: Stanford
By Shara Tonn

Researchers Create Versatile 3D Nanostructures Using DNA "Bricks"

ORIGINAL: Wyss Institute - Harvard
Nov 29, 2012 

Wyss Institute researchers have created more than 100 three-dimensional nanostructures using DNA building blocks that function like Lego® bricks. This animation illustrates the process used to build these structures.

New method greatly expands repertoire of nanobiotechnology applications in medicine and beyond.

Wyss Institute researchers have created more than 100 three-dimensional nanostructures using DNA building blocks that function like Lego® bricks. This video illustrates how DNA is used to build these structures.

Boston, MA -- Researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University have created more than 100 three-dimensional (3D) nanostructures using DNA building blocks that function like Lego® bricks -- a major advance from the two-dimensional (2D) structures the same team built a few months ago.

In effect, the advance means researchers just went from being able to build a flat wall of Legos®, to building a house. The new method, featured as a cover research article in the 30 November issue of Science, is the next step toward using DNA nanotechnologies for more sophisticated applications than ever possible before, such as "smart" medical devices that target drugs selectively to disease sites, programmable imaging probes, templates for precisely arranging inorganic materials in the manufacturing of next generation computer circuits, and more.

Computer-generated 3D models (left) and corresponding 2D projection microscopy images (right) of nanostructures self-assembled from synthetic DNA strands called DNA bricks. A master DNA brick collection defines a 25-nanometer cubic "molecular canvas" with 1000 voxels. By selecting subsets of bricks from this canvas, Ke et al. constructed a panel of 102 distinct shapes exhibiting sophisticated surface features as well as intricate interior cavities and tunnels. These nanostructures may enable diverse applications ranging from medicine to nanobiotechnology and electronics. [Image Credit: Yonggang Ke, Wyss Institute, Harvard University.]

The nanofabrication technique, called "DNA-brick self-assembly," uses short, synthetic strands of DNA that work like interlocking Lego® bricks. It capitalizes on the ability to program DNA to form into predesigned shapes thanks to the underlying "recipe" of DNA base pairs: A (adenosine) only binds to T (thymine) and C (cytosine) only binds to G (guanine).

Earlier this year, the Wyss team reported in Nature how they could create a collection of 2D shapes by stacking one DNA brick (42 bases in length) upon another.

But there's a "twist" in the new method required to build in 3D.
The trick is to start with an even smaller DNA brick (32 bases in length), which changes the orientation of every matched-up pair of bricks to a 90 degree angle -- giving every two Legos® a 3D shape. In this way, the team can use these bricks to build "out" in addition to "up," and eventually form 3D structures, such as a 25-nanometer solid cube containing hundreds of bricks. The cube becomes a "master" DNA "molecular canvas"; in this case, the canvas was comprised of 1000 so-called "voxels," which correspond to eight base-pairs and measure about 2.5 nanometers in size - meaning this is architecture at its tiniest.

The master canvas is where the modularity comes in: by simply selecting subsets of specific DNA bricks from the large cubic structure, the team built 102 3D structures with sophisticated surface features, as well as intricate interior cavities and tunnels. "This is a simple, versatile and robust method," says Peng Yin, Ph.D., Wyss core faculty member and senior author on the study.

The DNA-brick technique capitalizes on the ability of DNA strands to selectively attach to other strands, thanks to the underlying "recipe" of DNA base pairs. This animation shows how the DNA strands self-assemble to build a structure.  View Animation above

Another method used to build 3D structures, called DNA origami, is tougher to use to build complex shapes, Yin said, because it relies on a long "scaffold" strand of DNA that folds to interact with hundreds of shorter "staple" strands - and each new shape requires a new scaffold routing strategy and hence new staples. In contrast, the DNA brick method does not use any scaffold strand and therefore has a modular architecture; each brick can be added or removed independently.

"We are moving at lightning speed in our ability to devise ever more powerful ways to use biocompatible DNA molecules as structural building blocks for nanotechnology, which could have great value for medicine as well as non-medical applications," says Wyss Institute Founding Director Don Ingber, M.D., Ph.D.

The research team led by Yin, who is also an assistant professor of systems biology at Harvard Medical School (HMS), included Wyss Postdoctoral Fellow Yonggang Ke, Ph.D., and Wyss Graduate Student Luvena Ong. Another contributor was Wyss Core Faculty member William Shih, Ph.D., who also holds appointments at HMS and the Dana-Farber Cancer Institute. Visit Molecular-systems.net to learn more about the team's work.

The research was supported by the Office of Naval Research, the Army Research Office, the National Science Foundation, the National Institutes of Health, and the Wyss Institute for Biologically Inspired Engineering at Harvard University.

For more information, contact Kristen Kusek
Kristen.kusek@wyss.harvard.edu
+1 617-432-8266

###

About the Wyss Institute for Biologically Inspired Engineering at Harvard University
The Wyss Institute for Biologically Inspired Engineering at Harvard University (http://wyss.harvard.edu) uses Nature's design principles to develop bioinspired materials and devices that will transform medicine and create a more sustainable world. Working as an alliance among Harvard's Schools of Medicine, Engineering, and Arts & Sciences, and in partnership with Beth Israel Deaconess Medical Center, Brigham and Women's Hospital, Boston Children's Hospital, Dana Farber Cancer Institute, Massachusetts General Hospital, the University of Massachusetts Medical School, Spaulding Rehabilitation Hospital, Boston University and Tufts University, the Institute crosses disciplinary and institutional barriers to engage in high-risk research that leads to transformative technological breakthroughs. By emulating Nature's principles for self-organizing and self-regulating, Wyss researchers are developing innovative new engineering solutions for healthcare, energy, architecture, robotics, and manufacturing. These technologies are translated into commercial products and therapies through collaborations with clinical investigators, corporate alliances, and new start-ups. The Wyss Institute was recently awarded the prestigious World Technology Network award for innovation in biotechnology.

Nanoparticles deliver cargo inside mitochondria

ORIGINAL: Foresight Institute

Shanta Dhar, right, an assistant professor of chemistry in the UGA Franklin College of Arts and Sciences, and doctoral student Sean Marrache have fabricated nanoparticles that boost the effectiveness of drugs by delivering them to the mitochondria of cells (credit: University of Georgia).

Targeted drug delivery is one of the most important contributions of current and near-term nanotechnology to medicine. New research shows that specifically targeting one component of the cell makes nanoparticle-mediated drug delivery much more effective for a variety of applications. A hat tip to KurzweilAI.net for reprinting this University of Georgia news release “UGA researchers boost efficacy of drugs by using nanoparticles to target ‘powerhouse of cells’“:

Nanoparticles have shown great promise in the targeted delivery of drugs to cells, but researchers at the University of Georgia have refined the drug delivery process further by using nanoparticles to deliver drugs to a specific organelle within cells.

By targeting mitochondria, often called “the powerhouse of cells,” the researchers increased the effectiveness of mitochondria-acting therapeutics used to treat cancer, Alzheimer’s disease and obesity in studies conducted with cultured cells.

“The mitochondrion is a complex organelle that is very difficult to reach, but these nanoparticles are engineered so that they do the right job in the right place,” said senior author Shanta Dhar, an assistant professor of chemistry in the UGA Franklin College of Arts and Sciences.

Dhar and her co-author, doctoral student Sean Marrache, used a biodegradable, FDA-approved polymer to fabricate their nanoparticles and then used the particles to encapsulate and test drugs that treat a variety of conditions. Their results were published this week in early edition of the journal Proceedings of the National Academy of Sciences [abstract].

To test the effectiveness of their drug targeting system against cancer, they encapsulated the drug lonidamine, which works by inhibiting energy production in the mitochondria, and, separately, a form of the antioxidant vitamin E. They then treated cultured cancer cells and found that mitochondrial targeting increased the effectiveness of the drugs by more than 100 times when compared to the drugs alone and by five times when compared to the delivery of drugs with nanoparticles that target the outside of cells.

Similarly, the compound curcumin has shown promise in inhibiting formation of the amyloid plaques that are a hallmark of Alzheimer’s disease, but it quickly degrades in the presence of light and is broken down rapidly by the body. By encapsulating curcumin in the mitochondria-targeting nanoparticles, however, the researchers were able to restore the ability of brain cells in culture to survive despite the presence of a compound that encourages plaque formation. Nearly 100 percent of the cells treated with the mitochondria-targeting nanoparticles survived in the presence of the plaque-inducing compound, compared to 67 percent of cells treated with free curcumin and 70 percent of cells treated with nanoparticles that target the outside of cells.

Finally, the researchers encapsulated the obesity drug 2,4-DNP—which works by making energy production in the mitochondria less efficient—in their nanoparticles and found that it reduced the production of fat by cultured cells known as preadipocytes by 67 percent compared to cells treated with the drug alone and by 61 percent of cells treated with nanoparticles that target the outside of cells.

“A lot of diseases are associated with dysfunctional mitochondria, but many of the drugs that act on the mitochondria can’t get there,” Marrache said. “Rather than try to alter the drugs, which can reduce their effectiveness, we encapsulate them in these nanoparticles and precisely deliver them to the mitochondria.”

Dhar said that getting drugs to the mitochondria is no simple feat. Upon entering cells, nanoparticles enter a sorting center known as the endosome. The first thing Dhar and Marrache had to demonstrate was that the nanoparticles escape from the endosome and don’t end up in the cells’ disposal center, the lysosome.

The mitochondria itself is protected by two membranes separated by an interstitial space. The outer membrane only permits molecules of a certain size to pass through, while the inner membrane only permits molecules of a given range of charges to pass. The researchers constructed a library of nanoparticles and tested them until they identified the optimum size range—64 to 80 nanometers, or approximately 1,000 times finer than the width of a human hair—and an optimum surface charge, plus 34 millivolts.

Dhar notes the components they used to create the nanoparticles are FDA approved and that their methods are highly reproducible and therefore have the potential to be translated into clinical settings. The researchers are currently testing their targeted delivery system in rodents and say that preliminary results are promising.

“Mitochondrial dysfunctions cause many disorders in humans,” Dhar said, ” so there are several potential applications for this delivery system.”

Subject to the usual caveat that these nanoparticles are still in an early stage of testing, having been tested only in cell culture, it is remarkable that such effective targeting to reach the matrix of the mitochondria was achieved by the relatively crude strategy of optimizing only particle size and surface charge through engineering polymer composition. So success was achieved through clever application of biological knowledge more than through sophisticated atomically precise construction. It will be fascinating to watch the evolution of this technology as ever more sophisticated construction leads to increasing effectiveness. While we are waiting, this targeting of drug delivery to mitochondria is likely to be especially helpful because so many pathologies seem rooted in imperfections and consequences of the symbiosis that led to eukaryotic cells, and all complex life on Earth, nearly two billion years ago.

—James Lewis, PhD

A shiny new tool for imaging biomolecules

ORIGINAL: EurekAlert

Berkeley Lab researchers embed artificial membranes with billions of nanoantennas for enhanced optical studies

Gold triangle nanoparticles paired tip-to-tip in a bow-tie formation, serve as optical antennas. When a protein (green) bound to a fluorescently labeled SOS-catalyst passes through the the gaps between opposing tips of the triangles (plasmonic hot spots) fluorescence is amplified. Credit: (Image by Groves, et. al., Berkeley Lab) Usage Restrictions: courtesy of Jay Groves, Berkeley Lab, UC Berkeley, HHMI

At the heart of the immune system that protects our bodies from disease and foreign invaders is a vast and complex communications network involving millions of cells, sending and receiving chemical signals that can mean life or death. At the heart of this vast cellular signaling network are interactions between billions of proteins and other biomolecules. These interactions, in turn, are greatly influenced by the spatial patterning of signaling and receptor molecules. The ability to observe signaling spatial patterns in the immune and other cellular systems as they evolve, and to study the impact on molecular interactions and, ultimately, cellular communication, would be a critical tool in the fight against immunological and other disorders that lead to a broad range of health problems including cancer. Such a tool is now at hand.

An array of gold nanoparticles in the shape of triangles that are paired in a tip-to-tip formation, like a bow-tie, can serve as optical antennas, capturing and concentrating light waves into well-defined hot spots, where the plasmonic effect is greatly amplified. Credit: (Image by Groves, et. al., Berkeley Lab). Usage Restrictions: courtesy of Jay Groves, Berkeley Lab, UC Berkeley, HHMI. 

Researchers with the U.S. Department of Energy (DOE)'s Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkeley, have developed the first practical application of optical nanoantennas in cell membrane biology. A scientific team led by chemist Jay Groves has developed a technique for lacing artificial lipid membranes with billions of gold "bowtie" nanoantennas. Through the phenomenon known as "plasmonics," these nanoantennas can boost the intensity of a fluorescent or Raman optical signal from a protein passing through a plasmonic "hot-spot" tens of thousands of times without the protein ever being touched.

"Our technique is minimally invasive since enhancement of optical signals is achieved without requiring the molecules to directly interact with the nanoantenna," Groves says. "This is an important improvement over methods that rely on adsorption of molecules directly onto antennas where their structure, orientation, and behavior can all be altered."

Groves holds joint appointments with Berkeley Lab's Physical Biosciences Division and UC Berkeley's Chemistry Department, and is also a Howard Hughes Medical Institute investigator. He is the corresponding author of a paper that reports these results in the journal NanoLetters. The paper is titled "Single Molecule Tracking on Supported Membranes with Arrays of Optical Nanoantennas." Co-authoring the paper were Theo Lohmuller, Lars Iversen, Mark Schmidt, Christopher Rhodes, Hsiung-Lin Tu and Wan-Chen Lin.

Jay Groves is a chemist who holds appointments with Berkeley Lab, UC Berkeley and HHMI. Credit: (Photo by Roy Kaltschmidt, Berkeley Lab).