Tag Archives: DNA

Doing math in a test tube using analog DNA

Basically, scientists at Duke University (US) have created an analog computer at the nanoscale, which can perform basic arithmetic. From an Aug. 23, 2016 news item on ScienceDaily,

Often described as the blueprint of life, DNA contains the instructions for making every living thing from a human to a house fly.

But in recent decades, some researchers have been putting the letters of the genetic code to a different use: making tiny nanoscale computers.

In a new study, a Duke University team led by professor John Reif created strands of synthetic DNA that, when mixed together in a test tube in the right concentrations, form an analog circuit that can add, subtract and multiply as they form and break bonds.

Rather than voltage, DNA circuits use the concentrations of specific DNA strands as signals.

An Aug. 23, 2016 Duke University news release (also on EurekAlert), which originated the news item, describes how most DNA-based circuits operate and what makes the one from Duke different,

Other teams have designed DNA-based circuits that can solve problems ranging from calculating square roots to playing tic-tac-toe. But most DNA circuits are digital, where information is encoded as a sequence of zeroes and ones.

Instead, the new Duke device performs calculations in an analog fashion by measuring the varying concentrations of specific DNA molecules directly, without requiring special circuitry to convert them to zeroes and ones first.

Unlike the silicon-based circuits used in most modern day electronics, commercial applications of DNA circuits are still a long way off, Reif said.

For one, the test tube calculations are slow. It can take hours to get an answer.

“We can do some limited computing, but we can’t even begin to think of competing with modern-day PCs or other conventional computing devices,” Reif said.

But DNA circuits can be far tinier than those made of silicon. And unlike electronic circuits, DNA circuits work in wet environments, which might make them useful for computing inside the bloodstream or the soupy, cramped quarters of the cell.

The technology takes advantage of DNA’s natural ability to zip and unzip to perform computations. Just like Velcro and magnets have complementary hooks or poles, the nucleotide bases of DNA pair up and bind in a predictable way.

The researchers first create short pieces of synthetic DNA, some single-stranded and some double-stranded with single-stranded ends, and mix them in a test tube.

When a single strand encounters a perfect match at the end of one of the partially double-stranded ones, it latches on and binds, displacing the previously bound strand and causing it to detach, like someone cutting in on a dancing couple.

The newly released strand can in turn pair up with other complementary DNA molecules downstream in the circuit, creating a domino effect.

The researchers solve math problems by measuring the concentrations of specific outgoing strands as the reaction reaches equilibrium.

To see how their circuit would perform over time as the reactions proceeded, Reif and Duke graduate student Tianqi Song used computer software to simulate the reactions over a range of input concentrations. They have also been testing the circuit experimentally in the lab.

Besides addition, subtraction and multiplication, the researchers are also designing more sophisticated analog DNA circuits that can do a wider range of calculations, such as logarithms and exponentials.

Conventional computers went digital decades ago. But for DNA computing, the analog approach has its advantages, the researchers say. For one, analog DNA circuits require fewer strands of DNA than digital ones, Song said.

Analog circuits are also better suited for sensing signals that don’t lend themselves to simple on-off, all-or-none values, such as vital signs and other physiological measurements involved in diagnosing and treating disease.

The hope is that, in the distant future, such devices could be programmed to sense whether particular blood chemicals lie inside or outside the range of values considered normal, and release a specific DNA or RNA — DNA’s chemical cousin — that has a drug-like effect.

Reif’s lab is also beginning to work on DNA-based devices that could detect molecular signatures of particular types of cancer cells, and release substances that spur the immune system to fight back.

“Even very simple DNA computing could still have huge impacts in medicine or science,” Reif said.

Here’s a link to and a citation for the paper,

Analog Computation by DNA Strand Displacement Circuits by Tianqi Song, Sudhanshu Garg, Reem Mokhtar, Hieu Bui, and John Reif. ACS Synth. Biol., 2016, 5 (8), pp 898–912 DOI: 10.1021/acssynbio.6b00144 Publication Date (Web): July 01, 2016

Copyright © 2016 American Chemical Society

This paper is behind a paywall.

Graphene ribbons in solution bending and twisting like DNA

An Aug. 15, 2016 news item on ScienceDaily announces research into graphene nanoribbons and their DNA (deoxyribonucleic acid)-like properties,

Graphene nanoribbons (GNRs) bend and twist easily in solution, making them adaptable for biological uses like DNA analysis, drug delivery and biomimetic applications, according to scientists at Rice University.

Knowing the details of how GNRs behave in a solution will help make them suitable for wide use in biomimetics, according to Rice physicist Ching-Hwa Kiang, whose lab employed its unique capabilities to probe nanoscale materials like cells and proteins in wet environments. Biomimetic materials are those that imitate the forms and properties of natural materials.

An Aug. 15, 2016 Rice University (Texas, US) news release (also on EurekAlert), which originated the news item, describes the ribbons and the research in more detail,

Graphene nanoribbons can be thousands of times longer than they are wide. They can be produced in bulk by chemically “unzipping” carbon nanotubes, a process invented by Rice chemist and co-author James Tour and his lab.

Their size means they can operate on the scale of biological components like proteins and DNA, Kiang said. “We study the mechanical properties of all different kinds of materials, from proteins to cells, but a little different from the way other people do,” she said. “We like to see how materials behave in solution, because that’s where biological things are.” Kiang is a pioneer in developing methods to probe the energy states of proteins as they fold and unfold.

She said Tour suggested her lab have a look at the mechanical properties of GNRs. “It’s a little extra work to study these things in solution rather than dry, but that’s our specialty,” she said.

Nanoribbons are known for adding strength but not weight to solid-state composites, like bicycle frames and tennis rackets, and forming an electrically active matrix. A recent Rice project infused them into an efficient de-icer coating for aircraft.

But in a squishier environment, their ability to conform to surfaces, carry current and strengthen composites could also be valuable.

“It turns out that graphene behaves reasonably well, somewhat similar to other biological materials. But the interesting part is that it behaves differently in a solution than it does in air,” she said. The researchers found that like DNA and proteins, nanoribbons in solution naturally form folds and loops, but can also form helicoids, wrinkles and spirals.

Kiang, Wijeratne [Sithara Wijeratne, Rice graduate now a postdoctoral researcher at Harvard University] and Jingqiang Li, a co-author and student in the Kiang lab, used atomic force microscopy to test their properties. Atomic force microscopy can not only gather high-resolution images but also take sensitive force measurements of nanomaterials by pulling on them. The researchers probed GNRs and their precursors, graphene oxide nanoribbons.

The researchers discovered that all nanoribbons become rigid under stress, but their rigidity increases as oxide molecules are removed to turn graphene oxide nanoribbons into GNRs. They suggested this ability to tune their rigidity should help with the design and fabrication of GNR-biomimetic interfaces.

“Graphene and graphene oxide materials can be functionalized (or modified) to integrate with various biological systems, such as DNA, protein and even cells,” Kiang said. “These have been realized in biological devices, biomolecule detection and molecular medicine. The sensitivity of graphene bio-devices can be improved by using narrow graphene materials like nanoribbons.”

Wijeratne noted graphene nanoribbons are already being tested for use in DNA sequencing, in which strands of DNA are pulled through a nanopore in an electrified material. The base components of DNA affect the electric field, which can be read to identify the bases.

The researchers saw nanoribbons’ biocompatibility as potentially useful for sensors that could travel through the body and report on what they find, not unlike the Tour lab’s nanoreporters that retrieve information from oil wells.

Further studies will focus on the effect of the nanoribbons’ width, which range from 10 to 100 nanometers, on their properties.

Here’s a link to and a citation for the paper,

Detecting the Biopolymer Behavior of Graphene Nanoribbons in Aqueous Solution by Sithara S. Wijeratne, Evgeni S. Penev, Wei Lu, Jingqiang Li, Amanda L. Duque, Boris I. Yakobson, James M. Tour, & Ching-Hwa Kiang. Scientific Reports 6, Article number: 31174 (2016)  doi:10.1038/srep31174 Published online: 09 August 2016

This paper is open access.

Movies and science, science, science (Part 1 of 2)

In the last few years, there’s been a veritable plethora of movies (and television shows in Canada and the US) that are about science and technology or have a significant  component or investigate the social impact. The trend does not seem to be slowing.

This first of two parts features the film, *Hidden* Figures, and a play being turned into a film, Photograph 51. The second part features the evolving Theranos story and plans to turn it into a film, The Man Who Knew Infinity, a film about an Indian mathematician, the science of the recent all woman Ghostbusters, and an ezine devoted to science films.

For the following movie tidbits, I have David Bruggeman to thank.

Hidden Figures

From David’s June 21, 2016 post on his Pasco Phronesis blog (Note: A link has been removed),

Hidden Figures is a fictionalized treatment of the book of the same name written by Margot Lee Shetterly (and underwritten by the Sloan Foundation).  Neither the book nor the film are released yet.  The book is scheduled for a September release, and the film currently has a January release date in the U.S.

Both the film and the book focus on the story of African American women who worked as computers for the government at the Langley National Aeronautic Laboratory in Hampton, Virginia.  The women served as human computers, making the calculations NASA needed during the Space Race.  While the book features four women, the film is focused on three: Katherine Johnson (recipient of the Presidential Medal of Freedom), Dorothy Vaughan, and Mary Jackson.  They are played by, respectively, Taraji P. Henson, Octavia Spencer, and Janelle Monae.  Other actors in the film include Kevin Costner, Kirsten Dunst, Aldis Hodge, and Jim Parsons.  The film is directed by Theodore Melfi, and the script is by Allison Schroeder.

According to imdb.com, the movie’s release date is Dec. 25, 2016 (this could change again).

The history for ‘human computers’ stretches back to the 17th century, at least. From the Human Computer entry in Wikipedia (Note: Links have been removed),

The term “computer”, in use from the early 17th century (the first known written reference dates from 1613),[1] meant “one who computes”: a person performing mathematical calculations, before electronic computers became commercially available. “The human computer is supposed to be following fixed rules; he has no authority to deviate from them in any detail.” (Turing, 1950) Teams of people were frequently used to undertake long and often tedious calculations; the work was divided so that this could be done in parallel.

Prior to NASA, a team of women in the 19th century in the US were known as Harvard Computers (from the Wikipedia entry; Note: Links have been removed),

Edward Charles Pickering (director of the Harvard Observatory from 1877 to 1919) decided to hire women as skilled workers to process astronomical data. Among these women were Williamina Fleming, Annie Jump Cannon, Henrietta Swan Leavitt and Antonia Maury. This staff came to be known as “Pickering’s Harem” or, more respectfully, as the Harvard Computers.[1] This was an example of what has been identified as the “harem effect” in the history and sociology of science.

It seems that several factors contributed to Pickering’s decision to hire women instead of men. Among them was the fact that men were paid much more than women, so he could employ more staff with the same budget. This was relevant in a time when the amount of astronomical data was surpassing the capacity of the Observatories to process it.[2]

The first woman hired was Williamina Fleming, who was working as a maid for Pickering. It seems that Pickering was increasingly frustrated with his male assistants and declared that even his maid could do a better job. Apparently he was not mistaken, as Fleming undertook her assigned chores efficiently. When the Harvard Observatory received in 1886 a generous donation from the widow of Henry Draper, Pickering decided to hire more female staff and put Fleming in charge of them.[3]

While it’s not thrilling to find out that Pickering was content to exploit the women he was hiring, he deserves kudos for recognizing that women could do excellent work and acting on that recognition. When you consider the times, Pickering’s was an extraordinary act.

Getting back to Hidden Figures, an Aug.15, 2016 posting by Kathleen for Lainey Gossip celebrates the then newly released trailer for the movie,

If you’ve been watching the Olympics [Rio 2016], you know how much the past 10 days have been an epic display of #BlackGirlMagic. Fittingly, the trailer for Hidden Figures was released last night during Sunday’s Olympic coverage. It’s the story of three brilliant African American women, played by Taraji P Henson, Octavia Spencer and Janelle Monae, who made history by serving as the brains behind the NASA launch of astronaut John Glenn into orbit in 1962.

Three black women helped launch a dude into space in the 60s. AT NASA. Think about how America treated black women in the 60s. As Katherine Johnson, played by Taraji P Henson, jokes in the trailer, they were still sitting at the back of the bus. In 1962 Malcolm X said, “The most disrespected person in America is the Black woman, the most unprotected person in America is the Black woman. The most neglected person in America is the Black woman.” These women had to face that truth every day and they still rose to greatness. I’m obsessed with this story.

Overall, the trailer is good. I like the pace and the performances look strong. …

I’m most excited for Hidden Figures (as Lainey pointed out, this title is THE WORST) because black girls are being celebrated for their brains on screen. That is rare. When the trailer aired, my brother Sam texted me, “WHOA, a smart black girl movie!”

*ETA Sept. 5, 2016: Aran Shetterly contacted me to say this:

What you may not know is that the term “Hidden Figures” is a specific reference to flight science. It tested a pilot’s ability to pick out a simple figure from a set of more complex, difficult to see images. http://www.militaryaptitudetests.com/afoqt/

Thank you Mr. Shetterly!

Photograph 51 (the Rosalind Franklin story)

Also in David’s June 21, 2016 post is a mention of Photograph 51, a play and soon-to-be film about Rosalind Franklin, the discovery of the double helix, and a science controversy. I first wrote about Photograph 51 in a Jan. 16, 2012 posting (scroll down about 50% of the way) regarding an international script writing competition being held in Dublin, Ireland. At the time, I noted that Anna Ziegler’s play, Photograph 51 had won a previous competition cycle of the screenwriting competition. I wrote again about the play in a Sept. 2, 2015 posting about its London production (Sept. 5 – Nov. 21, 2015) featuring actress Nicole Kidman.

The versions of the Franklin story with which I’m familiar paint her as the wronged party, ignored and unacknowledged by the scientists (Francis, Crick, James Watson, and Maurice Wilkins) who got all the glory and the Nobel Prize. Stephen Curry in a Sept. 16, 2015 posting on the Guardian science blogs suggests the story may not be quite as simple as that (Note: A link has been removed),

Ziegler [Anna Ziegler, playwright] is up front in admitting that she has rearranged facts to suit the drama. This creates some oddities of chronology and motive for those familiar with the history. I know of no suggestion of romantic interest in Franklin from Wilkins, or of a separation of Crick from his wife in the aftermath of his triumph with Watson in solving the DNA structure. There is no mention in the play of the fact that Franklin published her work (and the famous photograph 51) in the journal Nature alongside Watson and Crick’s paper and one by Wilkins. Nor does the audience hear of the international recognition that Franklin enjoyed in her own right between 1953 and her untimely death in 1958, not just for her involvement in DNA, but also for her work on the structure of coal and of viruses.

Published long after her death, The Double Helix is widely thought to treat Franklin unfairly. In the minds of many she remains the wronged woman whose pioneering results were taken by others to solve DNA and win the Nobel prize. But the real story – many elements of which come across strongly in the play – is more complex*.

Franklin is a gifted experimentalist. Her key contributions to the discovery were in improving methods for taking X-ray pictures of and discovering the distinct A and B conformations of DNA. But it becomes clear that her methodical, meticulous approach to data analysis – much to Wilkins’ impotent frustration – eventually allows the Kings ‘team’ to be overtaken by the bolder, intuitive stratagem of Watson and Crick.

Curry’s piece is a good read and provides insight into the ways temperament affects how science is practiced.

Interestingly, there was a 1987 dramatization of the ‘double helix or life story’ (from the Life Story entry on Wikipedia; Note: Links have been removed),

The film tells the story of the rivalries of the two teams of scientists attempting to discover the structure of DNA. Francis Crick and James D. Watson at Cambridge University and Maurice Wilkins and Rosalind Franklin at King’s College London.

The film manages to convey the loneliness and competitiveness of scientific research but also educates the viewer as to how the structure of DNA was discovered. In particular, it explores the tension between the patient, dedicated laboratory work of Franklin and the sometimes uninformed intuitive leaps of Watson and Crick, all played against a background of institutional turf wars, personality conflicts and sexism. In the film Watson jokes, plugging the path of intuition: “Blessed are they who believed before there was any evidence.” The film also shows why Watson and Crick made their discovery, overtaking their competitors in part by reasoning from genetic function to predict chemical structure, thus helping to establish the then still-nascent field of molecular biology.

You can find out more about the stars, crew, and cast here on imdb.com

In addition to Life Story, the dramatization is also sometimes titled as ‘The Race for the Double Helix’ or the ‘Double Helix’.

Getting back to Photograph 51 (the film), Michael Grandage who directed the stage play will also direct the film. Grandage just made his debut as a film director with ‘Genius’ starring Colin Firth and Jude Law. According to this June 23, 2016 review by Sarah on Laineygossip.com, he stumbled a bit by casting British and Australian actors as Americans,

The first hurdle to clear with Genius, the feature film debut of English theater director Michael Grandage, is that everyone is played by Brits and Aussies, and by “everyone” I mean some of the most towering figures of American literature. You cast the best actor for the role and a good actor can convince you they’re anyone, so it shouldn’t really matter, but there is something profoundly odd about watching a parade of Lit 101 All Stars appear on screen and struggle with American accents. …

That kind of casting should not be a problem with Photograph 51 where the action takes place with British personalities.

Part 2 is here.

*’Human’ corrected to ‘Hidden’ on Sept. 5, 2016.

DNA as a sensor

McMaster University (Ontario, Canada) researchers have developed a technique for using DNA (deoxyribonucleic acid) as a sensor according to a July 7, 2016 news item on ScienceDaily,

Researchers at McMaster University have established a way to harness DNA as the engine of a microscopic “machine” they can turn on to detect trace amounts of substances that range from viruses and bacteria to cocaine and metals.

“It’s a completely new platform that can be adapted to many kinds of uses,” says John Brennan, director of McMaster’s Biointerfaces Insitute and co-author of a paper in the journal Nature Communications that describes the technology. “These DNA nano-architectures are adaptable, so that any target should be detectable.”

A July 7, 2016 McMaster University news release (also on EurekAlert), which originated the news item, expands on the theme,

DNA is best known as a genetic material, but is also a very programmable molecule that lends itself to engineering for synthetic applications.

The new method shapes separately programmed pieces of DNA material into pairs of interlocking circles.

The first remains inactive until it is released by the second, like a bicycle wheel in a lock. When the second circle, acting as the lock, is exposed to even a trace of the target substance, it opens, freeing the first circle of DNA, which replicates quickly and creates a signal, such as a colour change.

“The key is that it’s selectively triggered by whatever we want to detect,” says Brennan, who holds the Canada Research Chair in Bioanalytical Chemistry and Biointerfaces. “We have essentially taken a piece of DNA and forced it to do something it was never designed to do. We can design the lock to be specific to a certain key. All the parts are made of DNA, and ultimately that key is defined by how we build it.”

The idea for the “DNA nanomachine” comes from nature itself, explains co-author Yingfu Li, who holds the Canada Research Chair in Nucleic Acids Research.

“Biology uses all kinds of nanoscale molecular machines to achieve important functions in cells,” Li says. “For the first time, we have designed a DNA-based nano-machine that is capable of achieving ultra-sensitive detection of a bacterial pathogen.”

The DNA-based nanomachine is being further developed into a user-friendly detection kit that will enable rapid testing of a variety of substances, and could move to clinical testing within a year.

Here’s a link to and a citation for the paper,

Programming a topologically constrained DNA nanostructure into a sensor by Meng Liu, Qiang Zhang, Zhongping Li, Jimmy Gu, John D. Brennan, & Yingfu Li. Nature Communications 7, Article number: 12074  doi:10.1038/ncomms12074 Published 23 June 2016

This paper is open access.

Viewing RNA (ribonucleic acid) more closely at the nanoscale with expansion microscopy (EXM) and off-the-shelf parts

A close cousin to DNA (deoxyribonucleic acid), RNA (ribonucleic acid) is a communicator according to a July 4, 2016 news item on ScienceDaily describing how a team at the Massachusetts Institute of Technology (MIT) managed to image RNA more precisely,

Cells contain thousands of messenger RNA molecules, which carry copies of DNA’s genetic instructions to the rest of the cell. MIT engineers have now developed a way to visualize these molecules in higher resolution than previously possible in intact tissues, allowing researchers to precisely map the location of RNA throughout cells.

Key to the new technique is expanding the tissue before imaging it. By making the sample physically larger, it can be imaged with very high resolution using ordinary microscopes commonly found in research labs.

“Now we can image RNA with great spatial precision, thanks to the expansion process, and we also can do it more easily in large intact tissues,” says Ed Boyden, an associate professor of biological engineering and brain and cognitive sciences at MIT, a member of MIT’s Media Lab and McGovern Institute for Brain Research, and the senior author of a paper describing the technique in the July 4, 2016 issue of Nature Methods.

A July 4, 2016 MIT news release (also on EurekAlert), which originated the news item, explains why scientists want a better look at RNA and how the MIT team accomplished the task,

Studying the distribution of RNA inside cells could help scientists learn more about how cells control their gene expression and could also allow them to investigate diseases thought to be caused by failure of RNA to move to the correct location.

Boyden and colleagues first described the underlying technique, known as expansion microscopy (ExM), last year, when they used it to image proteins inside large samples of brain tissue. In a paper appearing in Nature Biotechnology on July 4, the MIT team has now presented a new version of the technology that employs off-the-shelf chemicals, making it easier for researchers to use.

MIT graduate students Fei Chen and Asmamaw Wassie are the lead authors of the Nature Methods paper, and Chen and graduate student Paul Tillberg are the lead authors of the Nature Biotechnology paper.

A simpler process

The original expansion microscopy technique is based on embedding tissue samples in a polymer that swells when water is added. This tissue enlargement allows researchers to obtain images with a resolution of around 70 nanometers, which was previously possible only with very specialized and expensive microscopes. However, that method posed some challenges because it requires generating a complicated chemical tag consisting of an antibody that targets a specific protein, linked to both a fluorescent dye and a chemical anchor that attaches the whole complex to a highly absorbent polymer known as polyacrylate. Once the targets are labeled, the researchers break down the proteins that hold the tissue sample together, allowing it to expand uniformly as the polyacrylate gel swells.

In their new studies, to eliminate the need for custom-designed labels, the researchers used a different molecule to anchor the targets to the gel before digestion. This molecule, which the researchers dubbed AcX, is commercially available and therefore makes the process much simpler.

AcX can be modified to anchor either proteins or RNA to the gel. In the Nature Biotechnology study, the researchers used it to anchor proteins, and they also showed that the technique works on tissue that has been previously labeled with either fluorescent antibodies or proteins such as green fluorescent protein (GFP).

“This lets you use completely off-the-shelf parts, which means that it can integrate very easily into existing workflows,” Tillberg says. “We think that it’s going to lower the barrier significantly for people to use the technique compared to the original ExM.”

Using this approach, it takes about an hour to scan a piece of tissue 500 by 500 by 200 microns, using a light sheet fluorescence microscope. The researchers showed that this technique works for many types of tissues, including brain, pancreas, lung, and spleen.

Imaging RNA

In the Nature Methods paper, the researchers used the same kind of anchoring molecule but modified it to target RNA instead. All of the RNAs in the sample are anchored to the gel, so they stay in their original locations throughout the digestion and expansion process.

After the tissue is expanded, the researchers label specific RNA molecules using a process known as fluorescence in situ hybridization (FISH), which was originally developed in the early 1980s and is widely used. This allows researchers to visualize the location of specific RNA molecules at high resolution, in three dimensions, in large tissue samples.

This enhanced spatial precision could allow scientists to explore many questions about how RNA contributes to cellular function. For example, a longstanding question in neuroscience is how neurons rapidly change the strength of their connections to store new memories or skills. One hypothesis is that RNA molecules encoding proteins necessary for plasticity are stored in cell compartments close to the synapses, poised to be translated into proteins when needed.

With the new system, it should be possible to determine exactly which RNA molecules are located near the synapses, waiting to be translated.

“People have found hundreds of these locally translated RNAs, but it’s hard to know where exactly they are and what they’re doing,” Chen says. “This technique would be useful to study that.”

Boyden’s lab is also interested in using this technology to trace the connections between neurons and to classify different subtypes of neurons based on which genes they are expressing.

There’s a brief (30 secs.), silent video illustrating the work (something about a ‘Brainbow Hippocampus’) made available by MIT,

Here’s a link to and a citation for the paper,

Nanoscale imaging of RNA with expansion microscopy by Fei Chen, Asmamaw T Wassie, Allison J Cote, Anubhav Sinha, Shahar Alon, Shoh Asano, Evan R Daugharthy, Jae-Byum Chang, Adam Marblestone, George M Church, Arjun Raj, & Edward S Boyden.     Nature Methods (2016)  doi:10.1038/nmeth.3899 Published online 04 July 2016

This paper is behind a paywall.

DNA as a framework for rationally designed nanostructures

After publishing a June 15, 2016 post about taking DNA (deoxyribonucleic acid) beyond genetics, it seemed like a good to publish a companion piece featuring a more technical explanation of at least one way DNA might provide the base for living computers and robots. From a June 13, 2016 BrookHaven National Laboratory news release (also on EurekAlert),

A cube, an octahedron, a prism–these are among the polyhedral structures, or frames, made of DNA that scientists at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory have designed to connect nanoparticles into a variety of precisely structured three-dimensional (3D) lattices. The scientists also developed a method to integrate nanoparticles and DNA frames into interconnecting modules, expanding the diversity of possible structures.

These achievements, described in papers published in Nature Materials and Nature Chemistry, could enable the rational design of nanomaterials with enhanced or combined optical, electric, and magnetic properties to achieve desired functions.

“We are aiming to create self-assembled nanostructures from blueprints,” said physicist Oleg Gang, who led this research at the Center for Functional Nanomaterials (CFN), a DOE Office of Science User Facility at Brookhaven. “The structure of our nanoparticle assemblies is mostly controlled by the shape and binding properties of precisely designed DNA frames, not by the nanoparticles themselves. By enabling us to engineer different lattices and architectures without having to manipulate the particles, our method opens up great opportunities for designing nanomaterials with properties that can be enhanced by precisely organizing functional components. For example, we could create targeted light-absorbing materials that harness solar energy, or magnetic materials that increase information-storage capacity.”

The news release goes on to describe the frames,

Gang’s team has previously exploited DNA’s complementary base pairing–the highly specific binding of bases known by the letters A, T, G, and C that make up the rungs of the DNA double-helix “ladder”–to bring particles together in a precise way. Particles coated with single strands of DNA link to particles coated with complementary strands (A binds with T and G binds with C) while repelling particles coated with non-complementary strands.

They have also designed 3D DNA frames whose corners have single-stranded DNA tethers to which nanoparticles coated with complementary strands can bind. When the scientists mix these nanoparticles and frames, the components self-assemble into lattices that are mainly defined by the shape of the designed frame. The Nature Materials paper describes the most recent structures achieved using this strategy.

“In our approach, we use DNA frames to promote the directional interactions between nanoparticles such that the particles connect into specific configurations that achieve the desired 3D arrays,” said Ye Tian, lead author on the Nature Materials paper and a member of Gang’s research team. “The geometry of each particle-linking frame is directly related to the lattice type, though the exact nature of this relationship is still being explored.”

So far, the team has designed five polyhedral frame shapes–a cube, an octahedron, an elongated square bipyramid, a prism, and a triangular bypyramid–but a variety of other shapes could be created.

“The idea is to construct different 3D structures (buildings) from the same nanoparticle (brick),” said Gang. “Usually, the particles need to be modified to produce the desired structures. Our approach significantly reduces the structure’s dependence on the nature of the particle, which can be gold, silver, iron, or any other inorganic material.”

Nanoparticles (yellow balls) capped with short single-stranded DNA (blue squiggly lines) are mixed with polyhedral DNA frames (from top to bottom): cube, octahedron, elongated square bipyramid, prism, and triangular bipyramid. The frames' vertices are encoded with complementary DNA strands for nanoparticle binding. When the corresponding frames and particles mix, they form a framework. Courtesy of Brookhaven National Laboratory

Nanoparticles (yellow balls) capped with short single-stranded DNA (blue squiggly lines) are mixed with polyhedral DNA frames (from top to bottom): cube, octahedron, elongated square bipyramid, prism, and triangular bipyramid. The frames’ vertices are encoded with complementary DNA strands for nanoparticle binding. When the corresponding frames and particles mix, they form a framework. Courtesy of Brookhaven National Laboratory

There’s also a discussion about how DNA origami was used to design the frames,

To design the frames, the team used DNA origami, a self-assembly technique in which short synthetic strands of DNA (staple strands) are mixed with a longer single strand of biologically derived DNA (scaffold strand). When the scientists heat and cool this mixture, the staple strands selectively bind with or “staple” the scaffold strand, causing the scaffold strand to repeatedly fold over onto itself. Computer software helps them determine the specific sequences for folding the DNA into desired shapes.

The folding of the single-stranded DNA scaffold introduces anchoring points that contain free “sticky” ends–unpaired strings of DNA bases–where nanoparticles coated with complementary single-strand tethers can attach. These sticky ends can be positioned anywhere on the DNA frame, but Gang’s team chose the corners so that multiple frames could be connected.

For each frame shape, the number of DNA strands linking a frame corner to an individual nanoparticle is equivalent to the number of edges converging at that corner. The cube and prism frames have three strands at each corner, for example. By making these corner tethers with varying numbers of bases, the scientists can tune the flexibility and length of the particle-frame linkages.

The interparticle distances are determined by the lengths of the frame edges, which are tens of nanometers in the frames designed to date, but the scientists say it should be possible to tailor the frames to achieve any desired dimensions.

The scientists verified the frame structures and nanoparticle arrangements through cryo-electron microscopy (a type of microscopy conducted at very low temperatures) at the CFN and Brookhaven’s Biology Department, and x-ray scattering at the National Synchrotron Light Source II (NSLS-II), a DOE Office of Science User Facility at Brookhaven.

The team started with a relatively simple form (from the news release),

In the Nature Chemistry paper, Gang’s team described how they used a similar DNA-based approach to create programmable two-dimensional (2D), square-like DNA frames around single nanoparticles.

DNA strands inside the frames provide coupling to complementary DNA on the nanoparticles, essentially holding the particle inside the frame. Each exterior side of the frame can be individually encoded with different DNA sequences. These outer DNA strands guide frame-frame recognition and connection.

Gang likens these DNA-framed nanoparticle modules to Legos whose interactions are programmed: “Each module can hold a different kind of nanoparticle and interlock to other modules in different but specific ways, fully determined by the complementary pairing of the DNA bases on the sides of the frame.”

In other words, the frames not only determine if the nanoparticles will connect but also how they will connect. Programming the frame sides with specific DNA sequences means only frames with complementary sequences can link up.

Mixing different types of modules together can yield a variety of structures, similar to the constructs that can be generated from Lego pieces. By creating a library of the modules, the scientists hope to be able to assemble structures on demand.

Finally, the discussion turns to the assembly of multifuctional nanomaterials (from the news release),

The selectivity of the connections enables different types and sizes of nanoparticles to be combined into single structures.

The geometry of the connections, or how the particles are oriented in space, is very important to designing structures with desired functions. For example, optically active nanoparticles can be arranged in a particular geometry to rotate, filter, absorb, and emit light–capabilities that are relevant for energy-harvesting applications, such as display screens and solar panels.

By using different modules from the “library,” Gang’s team demonstrated the self-assembly of one-dimensional linear arrays, “zigzag” chains, square-shaped and cross-shaped clusters, and 2D square lattices. The scientists even generated a simplistic nanoscale model of Leonardo da Vinci’s Vitruvian Man.

“We wanted to demonstrate that complex nanoparticle architectures can be self-assembled using our approach,” said Gang.

Again, the scientists used sophisticated imaging techniques–electron and atomic force microscopy at the CFN and x-ray scattering at NSLS-II–to verify that their structures were consistent with the prescribed designs and to study the assembly process in detail.

“Although many additional studies are required, our results show that we are making advances toward our goal of creating designed matter via self-assembly, including periodic particle arrays and complex nanoarchitectures with freeform shapes,” said Gang. “Our approach is exciting because it is a new platform for nanoscale manufacturing, one that can lead to a variety of rationally designed functional materials.”

Here’s an image illustrating among other things da Vinci’s Vitruvian Man,

A schematic diagram (left) showing how a nanoparticle (yellow ball) is incorporated within a square-like DNA frame. The DNA strands inside the frame (blue squiggly lines) are complementary to the DNA strands on the nanoparticle; the colored strands on the outer edges of the frame have different DNA sequences that determine how the DNA-framed nanoparticle modules can connect. The architecture shown (middle) is a simplistic nanoscale representation of Leonardo da Vinci's Vitruvian Man, assembled from several module types. The scientists used atomic force microscopy to generate the high-magnification image of this assembly (right). Courtesy Brookhaven National Laboratory

A schematic diagram (left) showing how a nanoparticle (yellow ball) is incorporated within a square-like DNA frame. The DNA strands inside the frame (blue squiggly lines) are complementary to the DNA strands on the nanoparticle; the colored strands on the outer edges of the frame have different DNA sequences that determine how the DNA-framed nanoparticle modules can connect. The architecture shown (middle) is a simplistic nanoscale representation of Leonardo da Vinci’s Vitruvian Man, assembled from several module types. The scientists used atomic force microscopy to generate the high-magnification image of this assembly (right). Courtesy Brookhaven National Laboratory

I enjoy the overviews provided by various writers and thinkers in the field but it’s details such as these that are often most compelling to me.

Taking DNA beyond genetics with living computers and nanobots

You might want to keep a salt shaker with you while reading a June 7, 2016 essay by Matteo Palma (Queen Mary’s University of London) about nanotechnology and DNA on The Conversation website (h/t June 7, 2016 news item on Nanowerk).

This is not a ‘hype’ piece as Palma backs every claim with links to the research while providing a good overview of some very exciting work but the mood is a bit euphoric so you may want to keep the earlier mentioned salt shaker nearby.

Palma offers a very nice beginner introduction especially helpful for someone who only half-remembers their high school biology (from the June 7, 2016 essay)

DNA is one of the most amazing molecules in nature, providing a way to carry the instructions needed to create almost any lifeform on Earth in a microscopic package. Now scientists are finding ways to push DNA even further, using it not just to store information but to create physical components in a range of biological machines.

Deoxyribonucleic acid or “DNA” carries the genetic information that we, and all living organisms, use to function. It typically comes in the form of the famous double-helix shape, made up of two single-stranded DNA molecules folded into a spiral. Each of these is made up of a series of four different types of molecular component: adenine (A), guanine (G), thymine (T), and cytosine (C).

Genes are made up from different sequences of these building block components, and the order in which they appear in a strand of DNA is what encodes genetic information. But by precisely designing different A,G,T and C sequences, scientists have recently been able to develop new ways of folding DNA into different origami shapes, beyond the conventional double helix.

This approach has opened up new possibilities of using DNA beyond its genetic and biological purpose, turning it into a Lego-like material for building objects that are just a few billionths of a metre in diameter (nanoscale). DNA-based materials are now being used for a variety of applications, ranging from templates for electronic nano-devices, to ways of precisely carrying drugs to diseased cells.

He highlights some Canadian work,

Designing electronic devices that are just nanometres in size opens up all sorts of possible applications but makes it harder to spot defects. As a way of dealing with this, researchers at the University of Montreal have used DNA to create ultrasensitive nanoscale thermometers that could help find minuscule hotspots in nanodevices (which would indicate a defect). They could also be used to monitor the temperature inside living cells.

The nanothermometers are made using loops of DNA that act as switches, folding or unfolding in response to temperature changes. This movement can be detected by attaching optical probes to the DNA. The researchers now want to build these nanothermometers into larger DNA devices that can work inside the human body.

He also mentions the nanobots that will heal your body (according to many works of fiction),

Researchers at Harvard Medical School have used DNA to design and build a nanosized robot that acts as a drug delivery vehicle to target specific cells. The nanorobot comes in the form of an open barrel made of DNA, whose two halves are connected by a hinge held shut by special DNA handles. These handles can recognise combinations of specific proteins present on the surface of cells, including ones associated with diseases.

When the robot comes into contact with the right cells, it opens the container and delivers its cargo. When applied to a mixture of healthy and cancerous human blood cells, these robots showed the ability to target and kill half of the cancer cells, while the healthy cells were left unharmed.

Palma is describing a very exciting development and there are many teams worldwide working on ways to make drugs more effective and less side effect-ridden. However there does seem to be a bit of a problem with targeted drug delivery as noted in my April 27, 2016 posting,

According to an April 27, 2016 news item on Nanowerk researchers at the University of Toronto (Canada) along with their collaborators in the US (Harvard Medical School) and Japan (University of Tokyo) have determined that less than 1% of nanoparticle-based drugs reach their intended destination …

Less than 1%? Admittedly, nanoparticles are not the same as nanobots but the problem is in the delivery, from my April 27, 2016 posting,

… the authors argue that, in order to increase nanoparticle delivery efficiency, a systematic and coordinated long-term strategy is necessary. To build a strong foundation for the field of cancer nanomedicine, researchers will need to understand a lot more about the interactions between nanoparticles and the body’s various organs than they do today. …

I imagine nanobots will suffer a similar fate since the actual delivery mechanism to a targeted cell is still a mystery.

I quite enjoyed Palma’s essay and appreciated the links he provided. My only proviso, keep a salt shaker nearby. That rosy future is going take a while to get here.

The Leonardo Project and the master’s DNA (deoxyribonucleic acid)

I’ve never really understood the mania for digging up bodies of famous people in history and trying to ascertain how the person really died or what kind of diseases they may have had but the practice fascinates me. The latest famous person to be subjected to a forensic inquiry centuries after death is Leonardo da Vinci. A May 5, 2016 Human Evolution (journal) news release on EurekAlert provides details,

A team of eminent specialists from a variety of academic disciplines has coalesced around a goal of creating new insight into the life and genius of Leonardo da Vinci by means of authoritative new research and modern detective technologies, including DNA science.

The Leonardo Project is in pursuit of several possible physical connections to Leonardo, beaming radar, for example, at an ancient Italian church floor to help corroborate extensive research to pinpoint the likely location of the tomb of his father and other relatives. A collaborating scholar also recently announced the successful tracing of several likely DNA relatives of Leonardo living today in Italy (see endnotes).

If granted the necessary approvals, the Project will compare DNA from Leonardo’s relatives past and present with physical remnants — hair, bones, fingerprints and skin cells — associated with the Renaissance figure whose life marked the rebirth of Western civilization.

The Project’s objectives, motives, methods, and work to date are detailed in a special issue of the journal Human Evolution, published coincident with a meeting of the group hosted in Florence this week under the patronage of Eugenio Giani, President of the Tuscan Regional Council (Consiglio Regionale della Toscana).

The news release goes on to provide some context for the work,

Born in Vinci, Italy, Leonardo died in 1519, age 67, and was buried in Amboise, southwest of Paris. His creative imagination foresaw and described innovations hundreds of years before their invention, such as the helicopter and armored tank. His artistic legacy includes the iconic Mona Lisa and The Last Supper.

The idea behind the Project, founded in 2014, has inspired and united anthropologists, art historians, genealogists, microbiologists, and other experts from leading universities and institutes in France, Italy, Spain, Canada and the USA, including specialists from the J. Craig Venter Institute of California, which pioneered the sequencing of the human genome.

The work underway resembles in complexity recent projects such as the successful search for the tomb of historic author Miguel de Cervantes and, in March 2015, the identification of England’s King Richard III from remains exhumed from beneath a UK parking lot, fittingly re-interred 500 years after his death.

Like Richard, Leonardo was born in 1452, and was buried in a setting that underwent changes in subsequent years such that the exact location of the grave was lost.

If DNA and other analyses yield a definitive identification, conventional and computerized techniques might reconstruct the face of Leonardo from models of the skull.”

In addition to Leonardo’s physical appearance, information potentially revealed from the work includes his ancestry and additional insight into his diet, state of health, personal habits, and places of residence.

According to the news release, the researchers have an agenda that goes beyond facial reconstruction and clues about  ancestry and diet,

Beyond those questions, and the verification of Leonardo’s “presumed remains” in the chapel of Saint-Hubert at the Château d’Amboise, the Project aims to develop a genetic profile extensive enough to understand better his abilities and visual acuity, which could provide insights into other individuals with remarkable qualities.

It may also make a lasting contribution to the art world, within which forgery is a multi-billion dollar industry, by advancing a technique for extracting and sequencing DNA from other centuries-old works of art, and associated methods of attribution.

Says Jesse Ausubel, Vice Chairman of the Richard Lounsbery Foundation, sponsor of the Project’s meetings in 2015 and 2016: “I think everyone in the group believes that Leonardo, who devoted himself to advancing art and science, who delighted in puzzles, and whose diverse talents and insights continue to enrich society five centuries after his passing, would welcome the initiative of this team — indeed would likely wish to lead it were he alive today.”

The researchers aim to have the work complete by 2019,

In the journal, group members underline the highly conservative, precautionary approach required at every phase of the Project, which they aim to conclude in 2019 to mark the 500th anniversary of Leonardo’s death.

For example, one objective is to verify whether fingerprints on Leonardo’s paintings, drawings, and notebooks can yield DNA consistent with that extracted from identified remains.

Early last year, Project collaborators from the International Institute for Humankind Studies in Florence opened discussions with the laboratory in that city where Leonardo’s Adoration of the Magi has been undergoing restoration for nearly two years, to explore the possibility of analyzing dust from the painting for possible DNA traces. A crucial question is whether traces of DNA remain or whether restoration measures and the passage of time have obliterated all evidence of Leonardo’s touch.

In preparation for such analysis, a team from the J. Craig Venter Institute and the University of Florence is examining privately owned paintings believed to be of comparable age to develop and calibrate techniques for DNA extraction and analysis. At this year’s meeting in Florence, the researchers also described a pioneering effort to analyze the microbiome of a painting thought to be about five centuries old.

If human DNA can one day be obtained from Leonardo’s work and sequenced, the genetic material could then be compared with genetic information from skeletal or other remains that may be exhumed in the future.

Here’s a list of the participating organizations (from the news release),

  • The Institut de Paléontologie Humaine, Paris
  • The International Institute for Humankind Studies, Florence
  • The Laboratory of Molecular Anthropology and Paleogenetics, Biology Department, University of Florence
  • Museo Ideale Leonardo da Vinci, in Vinci, Italy
  • J. Craig Venter Institute, La Jolla, California
  • Laboratory of Genetic Identification, University of Granada, Spain
  • The Rockefeller University, New York City

You can find the special issue of Human Evolution (HE Vol. 31, 2016 no. 3) here. The introductory essay is open access but the other articles are behind a paywall.

Disinfectant for backyard pools could be key to new nanomaterials

Research from McGill University (Québec, Canada) focuses on cyanuric acid, one of the chemicals used to disinfect backyard pools. according to a March 1, 2016 McGill University news release (received by email; it can also be found in a March 1, 2016 news item on Nanowerk *and on EurekAlert*),

Cyanuric acid is commonly used to stabilize chlorine in backyard pools; it binds to free chlorine and releases it slowly in the water. But researchers at McGill University have now discovered that this same small, inexpensive molecule can also be used to coax DNA into forming a brand new structure: instead of forming the familiar double helix, DNA’s nucleobases — which normally form rungs in the DNA ladder — associate with cyanuric acid molecules to form a triple helix.

The discovery “demonstrates a fundamentally new way to make DNA assemblies,” says Hanadi Sleiman, Canada Research Chair in DNA Nanoscience at McGill and senior author of the study, published in Nature Chemistry. “This concept may apply to many other molecules, and the resulting DNA assemblies could have applications in a range of technologies.”

The DNA alphabet, composed of the four letters A, T, G and C, is the underlying code that gives rise to the double helix famously discovered by Watson and Crick more than 60 years ago. The letters, or bases, of DNA can also interact in other ways to form a variety of DNA structures used by scientists in nanotechnology applications – quite apart from DNA’s biological role in living cells.

For years, scientists have sought to develop a larger, designer alphabet of DNA bases that would enable the creation of more DNA structures with unique, new properties. For the most part, however, devising these new molecules has involved costly and complex procedures.

The road to the McGill team’s discovery began some eight years ago, when Sleiman mentioned to others in her lab that cyanuric acid might be worth experimenting with because of its properties. The molecule has three faces with the same binding features as thymine (T in the DNA alphabet), the natural complement to adenine (A).  “One of my grad students tried it,” she recalls, “and came back and said he saw fibres” through an atomic force microscope.

The researchers later discovered that these fibres have a unique underlying structure. Cyanuric acid is able to coax strands composed of adenine bases into forming a novel motif in DNA assembly. The adenine and cyanuric acid units associate into flower-like rosettes; these form the cross-section of a triple helix.  The strands then combine to form long fibres.

“The nanofibre material formed in this way is easy to access, abundant and highly structured,” says Nicole Avakyan, a PhD student in Sleiman’s lab and first author of the study. “With further development, we can envisage a variety of applications of this material, from medicinal chemistry to tissue engineering and materials science.”

Here’s a link to and a citation for the paper,

Reprogramming the assembly of unmodified DNA with a small molecule by Nicole Avakyan, Andrea A. Greschner, Faisal Aldaye, Christopher J. Serpell, Violeta Toader,    Anne Petitjean, & Hanadi F. Sleiman. Nature Chemistry (2016) doi:10.1038/nchem.2451 Published online 22 February 2016

This paper is behind a paywall.

*’also on EurekAlert’ added on March 2, 2016.

Shape-shifting nanoparticles for better chemotherapy from the University of Toronto (Canada)

A research team from the University of Toronto and its shape-shifting nanoparticles are being touted in a Feb. 19, 2016 news item on Nanowerk,

Chemotherapy isn’t supposed to make your hair fall out — it’s supposed to kill cancer cells. A new molecular delivery system created at U of T [University of Toronto] Engineering could help ensure that chemotherapy drugs get to their target while minimizing collateral damage.

Many cancer drugs target fast-growing cells. Injected into a patient, they swirl around in the bloodstream acting on fast-growing cells wherever they find them. That includes tumours, but unfortunately also hair follicles, the lining of your digestive system, and your skin.

U of T Engineering Professor Warren Chan has spent the last decade figuring out how to deliver chemotherapy drugs into tumours — and nowhere else. Now his lab has designed a set of nanoparticles attached to strands of DNA that can change shape to gain access to diseased tissue.

A Feb. 18, 2016 University of Toronto news release (also on EurekAlert), which originated the news item, expands on the theme,

“Your body is basically a series of compartments,” says Chan. “Think of it as a giant house with rooms inside. We’re trying to figure out how to get something that’s outside, into one specific room. One has to develop a map and a system that can move through the house where each path to the final room may have different restrictions such as height and width.”

One thing we know about cancer: no two tumours are identical. Early-stage breast cancer, for example, may react differently to a given treatment than pancreatic cancer, or even breast cancer at a more advanced stage. Which particles can get inside which tumours depends on multiple factors such as the particle’s size, shape and surface chemistry.

Chan and his research group have studied how these factors dictate the delivery of small molecules and nanotechnologies to tumours, and have now designed a targeted molecular delivery system that uses modular nanoparticles whose shape, size and chemistry can be altered by the presence of specific DNA sequences.

“We’re making shape-changing nanoparticles,” says Chan. “They’re a series of building blocks, kind of like a LEGO set.” The component pieces can be built into many shapes, with binding sites exposed or hidden. They are designed to respond to biological molecules by changing shape, like a key fitting into a lock.

These shape-shifters are made of minuscule chunks of metal with strands of DNA attached to them. Chan envisions that the nanoparticles will float around harmlessly in the blood stream, until a DNA strand binds to a sequence of DNA known to be a marker for cancer. When this happens, the particle changes shape, then carries out its function: it can target the cancer cells, expose a drug molecule to the cancerous cell, tag the cancerous cells with a signal molecule, or whatever task Chan’s team has designed the nanoparticle to carry out.

“We were inspired by the ability of proteins to alter their conformation — they somehow figure out how to alleviate all these delivery issues inside the body,” says Chan. “Using this idea, we thought, ‘Can we engineer a nanoparticle to function like a protein, but one that can be programmed outside the body with medical capabilities?’”

Applying nanotechnology and materials science to medicine, and particularly to targeted drug delivery, is still a relatively new concept, but one Chan sees as full of promise. The real problem is how to deliver enough of the nanoparticles directly to the cancer to produce an effective treatment.

“Here’s how we look at these problems: it’s like you’re going to Vancouver from Toronto, but no one tells you how to get there, no one gives you a map, or a plane ticket, or a car — that’s where we are in this field,” he says. “The idea of targeting drugs to tumours is like figuring out how to go to Vancouver. It’s a simple concept, but to get there isn’t simple if not enough information is provided.”

“We’ve only scratched the surface of how nanotechnology ‘delivery’ works in the body, so now we’re continuing to explore different details of why and how tumours and other organs allow or block certain things from getting in,” adds Chan.

He and his group plan to apply the delivery system they’ve designed toward personalized nanomedicine — further tailoring their particles to deliver drugs to your precise type of tumour, and nowhere else.

Here are links to and citations for the team’s two published papers,

DNA-controlled dynamic colloidal nanoparticle systems for mediating cellular interaction by Seiichi Ohta, Dylan Glancy, Warren C. W. Chan. Science  19 Feb 2016: Vol. 351, Issue 6275, pp. 841-845 DOI: 10.1126/science.aad4925

Tailoring nanoparticle designs to target cancer based on tumor pathophysiology by Edward A. Sykes, Qin Dai, Christopher D. Sarsons, Juan Chen, Jonathan V. Rocheleau, David M. Hwang, Gang Zheng, David T. Cramb, Kristina D. Rinker, and Warren C. W. Chan. PNAS     doi: 10.1073/pnas.1521265113 published online Feb. 16, 2016.

Both papers are behind paywalls.