Making error-free DNA from RNA

For 3 billion years, one of the major carriers of information needed for life, RNA, has had a glitch that creates errors when making copies of genetic information. Researchers at The University of Texas at Austin have developed a fix that allows RNA to accurately proofread for the first time. The new discovery, published June 23 in the journal Science, will increase precision in genetic research and could dramatically improve medicine based on a person's genetic makeup.

Certain viruses called retroviruses can cause RNA to make copies of DNA, a process called reverse transcription. This process is notoriously prone to errors because an evolutionary ancestor of all viruses never had the ability to accurately copy genetic material.

The new innovation engineered at UT Austin is an enzyme that performs reverse transcription but can also "proofread," or check its work while copying genetic code. The enzyme allows, for the first time, for large amounts of RNA information to be copied with near perfect accuracy.

"We created a new group of enzymes that can read the genetic information inside living cells with unprecedented accuracy," says Jared Ellefson, a postdoctoral fellow in UT Austin's Center for Systems and Synthetic Biology. "Overlooked by evolution, our enzyme can correct errors while copying RNA."

Reverse transcription is mainly associated with retroviruses such as HIV. In nature, these viruses' inability to copy DNA accurately may have helped create variety in species over time, contributing to the complexity of life as we know it.

Since discovering reverse transcription, scientists have used it to better understand genetic information related to inheritable diseases and other aspects of human health. Still, the error-prone nature of existing RNA sequencing is a problem for scientists.

"With proofreading, our new enzyme increases precision and fidelity of RNA sequencing," says Ellefson. "Without the ability to faithfully read RNA, we cannot accurately determine the inner workings of cells. These errors can lead to misleading data in the research lab and potential misdiagnosis in the clinical lab."

Ellefson and the team of researchers engineered the new enzyme using directed evolution to train a high-fidelity (proofreading) DNA polymerase to use RNA templates. The new enzyme, called RTX, retains the highly accurate and efficient proofreading function, while copying RNA. Accuracy is improved at least threefold, and it may be up to 10 times as accurate. This new enzyme could enhance the methods used to read RNA from cells.

"As we move towards an age of personalized medicine where everyone's transcripts will be read out almost as easily as taking a pulse, the accuracy of the sequence information will become increasingly important," said Andy Ellington, a professor of molecular biosciences. "The significance of this is that we can now also copy large amounts of RNA information found in modern genomes, in the form of the RNA transcripts that encode almost every aspect of our physiology. This means that diagnoses made based on genomic information are far more likely to be accurate. "

Reference:

Synthetic evolutionary origin of a proofreading reverse transcriptase
Science  24 Jun 2016:
Vol. 352, Issue 6293, pp. 1590-1593
DOI: 10.1126/science.aaf5409


Nanotechnology World Association

Graphene patch could help patients manage diabetes

A wearable, graphene-based patch could one day maintain healthy blood glucose levels in people by measuring the sugar in sweat and then delivering the necessary dose of a diabetes drug through the skin (Nat. Nanotech. 2016, DOI:10.1038/nnano.2016.38).

The device takes scientists a step closer to the “coveted prize” in diabetes care: a noninvasive method to monitor and control blood glucose levels, writes Richard Guyof the University of Bath in a commentary about the new work.

Currently, most diabetic patients keep track of their blood glucose levels by pricking their fingers and testing a resulting droplet of blood. For people who must monitor their levels regularly, this can be a literal pain. “There are a lot of people who don’t like sticking things in their skin,” Guy says.

About 15 years ago, the Food & Drug Administration approved a noninvasive glucose-monitoring device called the GlucoWatch Biographer. Patients wore it on their wrists, and it extracted glucose from interstitial fluid in the skin using a small current. It didn’t catch on, in part because it wasn’t user friendly, Guy tells C&EN.

For the new patch, the researchers, led by Dae-Hyeong Kim of Seoul National University, decided to detect glucose in sweat because previous studies had shown that levels of the sugar in perspiration match those in blood. Other groups have also developed devices that can analyze biomolecules in sweat (C&EN, Feb. 1, 2016, page 11).

The new device uses layers of the fluoropolymer Nafion to absorb sweat and carry it toward the device’s sensors, which are built on modified graphene. The team doped the graphene with gold atoms and functionalized it with electrochemically active materials to enable reactions needed to detect glucose.

In the patch’s glucose sensors, the enzyme glucose oxidase reacts with the sugar and produces hydrogen peroxide, which, through an electrochemical reaction, extracts current from the doped graphene. This produces an electrical signal proportional to the amount of glucose present. The patch also contains pH and temperature sensors that help ensure that the glucose sensor’s signals accurately reflect the sugar’s concentration in sweat.

When two healthy volunteers wore the patch, the measured glucose levels—including spikes after meals—matched those from a commercial glucose meter. To monitor the levels, the patch sent its sensor signals to a device that analyzed them and then wirelessly relayed the data to a smartphone.

The drug delivery half of the patch consists of an array of 1-mm-tall polymer microneedles that pierce the skin. Each needle is made from a mixture of the diabetes drug metformin and a dissolvable polymer, polyvinyl pyrrolidone. And the needles are coated with a layer of tridecanoic acid. A gold and graphene mesh sits on top of the needle array and serves as a heater that can melt the coatings.

Once the tridecanoic acid melts, the needle dissolves in the skin and releases its drug payload.

When researchers applied just the drug-delivery component to the stomachs of diabetic mice, they could deliver enough metformin to lower the animal’s elevated blood glucose levels by more than 50% in 6 hours.

Guy thinks the sensor portion of the patch is closer to real-world use than the drug-delivery component. To make the drug-delivery system practical, he says, the researchers must make the microneedle array as small as possible. That means they must find a drug that’s effective at low doses.

As for the glucose-detection half of the device, Guy wonders how often a user would have to calibrate the sensors to ensure accurate readings.

Still, he calls the patch an impressive proof of concept.

University of Bath

Nature Materials: Smallest lattice structure worldwide

The smallest lattice in the world is visible under the microscope only. Struts and braces are 0.2 µm in diameter. Total size of the lattice is about 10 µm. Photo: J. Bauer / KIT

KIT scientists now present the smallest lattice structure made by man in the Nature Materials journal. Its struts and braces are made of glassy carbon and are less than 1 µm long and 200 nm in diameter. They are smaller than comparable metamaterials by a factor of 5. The small dimension results in so far unreached ratios of strength to density. Applications as electrodes, filters or optical components might be possible. (DOI: 10.1038/nmat4561)

"Lightweight construction materials, such as bones and wood, are found everywhere in nature," Dr.-Ing. Jens Bauer of Karlsruhe Institute of Technology (KIT), the first author of the study, explains. "They have a high load-bearing capacity and small weight and, hence, serve as models for mechanical metamaterials for technical applications."

Metamaterials are materials, whose structures of some micrometers (millionths of a meter) in dimension are planned and manufactured specifically for them to possess mechanical or optical properties that cannot be reached by unstructured solids. Examples are invisibility cloaks that guide light, sound or heat around objects, materials that counterintuitively react to pressure and shear (auxetic materials) or lightweight nanomaterials of high specific stability (force per unit area and density).

The smallest stable lattice structure worldwide presented now was produced by the established 3D laser lithography process at first. The desired structure of micrometer size is hardened in a photoresist by laser beams in a computer-controlled manner. However, resolution of this process is limited, such that struts of about 5 - 10 µm length and 1 µm in diameter can be produced only. In a subsequent step, the structure was therefore shrunk and vitrified by pyrolysis. For the first time, pyrolysis was used for manufacturing microstructured lattices. The object is exposed to temperatures of around 900°C in a vacuum furnace. As a result, chemical bonds reorient themselves. Except for carbon, all elements escape from the resist. The unordered carbon remains in the shrunk lattice structure in the form of glassy carbon. The resulting structures were tested for stability under pressure by the researchers.

"According to the results, load-bearing capacity of the lattice is very close to the theoretical limit and far above that of unstructured glassy carbon," Prof. Oliver Kraft, co-author of the study, reports. Until the end of last year, Kraft headed the Institute for Applied Materials of KIT. This year, he took over office as KIT Vice President for Research. "Diamond is the only solid having a higher specific stability."

Microstructured materials are often used for insulation or shock absorption. Open-pored materials may be used as filters in chemical industry. Metamaterials also have extraordinary optical properties that are applied in telecommunications. Glassy carbon is a high-technology material made of pure carbon. It combines glassy, ceramic properties with graphite properties and is of interest for use in electrodes of batteries or electrolysis systems.

KIT

Darwin on a chip

UT researchers develop (r)evolutionary circuits

Researchers of the MESA+ Institute for Nanotechnology and the CTIT Institute for ICT Research at the University of Twente in The Netherlands have demonstrated working electronic circuits that have been produced in a radically new way, using methods that resemble Darwinian evolution. The size of these circuits is comparable to the size of their conventional counterparts, but they are much closer to natural networks like the human brain. The findings promise a new generation of powerful, energy-efficient electronics, and have been published in the leading British journal Nature Nanotechnology.

One of the greatest successes of the 20th century has been the development of digital computers. During the last decades these computers have become more and more powerful by integrating ever smaller components on silicon chips. However, it is becoming increasingly hard and extremely expensive to continue this miniaturisation. Current transistors consist of only a handful of atoms. It is a major challenge to produce chips in which the millions of transistors have the same characteristics, and thus to make the chips operate properly. Another drawback is that their energy consumption is reaching unacceptable levels. It is obvious that one has to look for alternative directions, and it is interesting to see what we can learn from nature. Natural evolution has led to powerful ‘computers’ like the human brain, which can solve complex problems in an energy-efficient way. Nature exploits complex networks that can execute many tasks in parallel.

Moving away from designed circuits

The approach of the researchers at the University of Twente is based on methods that resemble those found in Nature. They have used networks of gold nanoparticles for the execution of essential computational tasks. Contrary to conventional electronics, they have moved away from designed circuits. By using 'designless' systems, costly design mistakes are avoided. The computational power of their networks is enabled by applying artificial evolution. This evolution takes less than an hour, rather than millions of years. By applying electrical signals, one and the same network can be configured into 16 different logical gates. The evolutionary approach works around - or can even take advantage of - possible material defects that can be fatal in conventional electronics.

Powerful and energy-efficient

It is the first time that scientists have succeeded in this way in realizing robust electronics with dimensions that can compete with commercial technology. According to prof. Wilfred van der Wiel, the realized circuits currently still have limited computing power. “But with this research we have delivered proof of principle: demonstrated that our approach works in practice. By scaling up the system, real added value will be produced in the future. Take for example the efforts to recognize patterns, such as with face recognition. This is very difficult for a regular computer, while humans and possibly also our circuits can do this much better."  Another important advantage may be that this type of circuitry uses much less energy, both in the production, and during use. The researchers anticipate a wide range of applications, for example in portable electronics and in the medical world.

University of Twente

A ‘printing press’ for nanoparticles

This new technique could facilitate use of gold nanoparticles in electronic, medical applications

Gold nanoparticles have unusual optical, electronic and chemical properties, which scientists are seeking to put to use in a range of new technologies, from nanoelectronics to cancer treatments.

Some of the most interesting properties of nanoparticles emerge when they are brought close together – either in clusters of just a few particles or in crystals made up of millions of them.  Yet particles that are just millionths of an inch in size are too small to be manipulated by conventional lab tools, so a major challenge has been finding ways to assemble these bits of gold while controlling the three-dimensional shape of their arrangement.

One approach that researchers have developed has been to use tiny structures made from synthetic strands of DNA to help organize nanoparticles. Since DNA strands are programmed to pair with other strands in certain patterns, scientists have attached individual strands of DNA to gold particle surfaces to create a variety of assemblies. But these hybrid gold-DNA nanostructures are intricate and expensive to generate, limiting their potential for use in practical materials. The process is similar, in a sense, to producing books by hand.

Enter the nanoparticle equivalent of the printing press. It’s efficient, re-usable and carries more information than previously possible. In results reported online in Nature Chemistry, researchers from McGill’s Department of Chemistry outline a procedure for making a DNA structure with a specific pattern of strands coming out of it; at the end of each strand is a chemical “sticky patch.”  When a gold nanoparticle is brought into contact to the DNA nanostructure, it sticks to the patches. The scientists then dissolve the assembly in distilled water, separating the DNA nanostructure into its component strands and leaving behind the DNA imprint on the gold nanoparticle. (See illustration.)

“These encoded gold nanoparticles are unprecedented in their information content,” says senior author Hanadi Sleiman, who holds the Canada Research Chair in DNA Nanoscience. “The DNA nanostructures, for their part, can be re-used, much like stamps in an old printing press.”

From stained glass to optoelectronics

Some of the properties of gold nanoparticles have been recognized for centuries.  Medieval artisans added gold chloride to molten glass to create the ruby-red colour in stained-glass windows – the result, as chemists figured out much later, of the light-scattering properties of tiny gold particles. 

Now, the McGill researchers hope their new production technique will help pave the way for use of DNA-encoded nanoparticles in a range of cutting-edge technologies. First author Thomas Edwardson says the next step for the lab will be to investigate the properties of structures made from these new building blocks. “In much the same way that atoms combine to form complex molecules, patterned DNA gold particles can connect to neighbouring particles to form well-defined nanoparticle assemblies.”

These could be put to use in areas including optoelectronic nanodevices and biomedical sciences, the researchers say. The patterns of DNA strands could, for example, be engineered to target specific proteins on cancer cells, and thus serve to detect cancer or to selectively destroy cancer cells. 

Financial support for the research was provided by the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation, the Centre for Self-Assembled Chemical Structures, the Canada Research Chairs Program and the Canadian Institutes of Health Research.

“Transfer of molecular recognition information from DNA nanostructures to gold nanoparticles,” Thomas G. W. Edwardson et al, Nature Chemistry, Jan. 4, 2016. DOI: 10.1038/nchem.2420
http://www.nature.com/nchem/journal/vaop/ncurrent/full/nchem.2420.html

McGill University

Nanotech weapon against chronic bacterial infections in hospitals

One of the scourges of infections in hospitals -- biofilms formed by bacteria that stick to each other on living tissue and medical instruments, making them harder to remove -- can be tricked into dispersing with the targeted application of nanoparticles and heat, researchers have found.

The University of New South Wales study, jointly led by Associate Professor Cyrille Boyer of the School of Chemical Engineering and deputy director of Australian Centre for NanoMedicine, appears in today's issue of Nature's open access journal Scientific Reports.

"Chronic biofilm-based infections are often extremely resistant to antibiotics and many other conventional antimicrobial agents, and have a high capacity to evade the body's immune system," said Boyer. "Our study points to a pathway for the non-toxic dispersal of biofilms in infected tissue, while also greatly improving the effect of antibiotic therapies." 

Biofilms have been linked to 80% of infections, forming on living tissues (eg. respiratory, gastrointestinal and urinary tracts, oral cavities, eyes, ears, wounds, heart and cervix) or dwelling in medical devices (eg. dialysis catheters, prosthetic implants and contact lenses).

The formation of biofilms is a growing and costly problem in hospitals, creating infections that are more difficult to treat -- leading to chronic inflammation, impaired wound healing, rapidly acquired antibiotic resistance and the spread of infectious embolisms in the bloodstream.

They also cause fouling and corrosion of wet surfaces, and the clogging of filtration membranes in sensitive equipment -- even posing a threat to public health by acting as reservoirs of pathogens in distribution systems for drinking water.

In general, bacteria have two life forms during growth and proliferation: planktonic, where bacteria exist as single, independent cells; or aggregated together in colonies as biofilms, where bacteria grow in a slime-like polymer matrix that protects them from the environment around them.

Acute infections mostly involve planktonic bacteria, which are usually treatable with antibiotics.

However, when bacteria have had enough time to form a biofilm -- within a human host or non-living material such as dialysis catheters -- an infection can often become untreatable and develop into a chronic state.

Although biofilms were first recognised in the 17th century, their importance was not realised until the 1990s, when it became clear that microbes exist in nature more often in colonies made up of lots of different microorganisms that adhere to surfaces through slime excreted by their inhabitants. Thus began a global race to understand biofilms, at a time when it was also realised they were responsible for the majority of chronic infections.

The discovery of how to dislodge biofilms by the UNSW team - jointly led by Dr Nicolas Barraud, formerly of UNSW and now at France's Institut Pasteur -- was made using the opportunistic human pathogen Pseudomonas aeruginosa. This is a model organism whose response to the technique the researchers believe will apply to most other bacteria.

When biofilms want to colonise a new site, they disperse into individual cells, reducing the protective action of the biofilm. It is this process the UNSW team sought to trigger, making the bacteria again susceptible to antimicrobial agents.

The UNSW team found that by injecting iron oxide nanoparticles into the biofilms, and using an applied magnetic field to heat them -- which induces local hyperthermia through raising the temperature by 5°C or more - the biofilms were triggered into dispersing.

They achieved this using iron oxide nanoparticles coated with polymers that help stabilise and maintain the nanoparticles in a dispersed state, making them an ideal non-toxic tool for treating biofilm infections.

"The use of these polymer-coated iron oxide nanoparticles to disperse biofilms may have broad applications across a range of clinical and industrial settings," said Boyer, who in October was named Physical Scientist of the Year in Australia's Prime Minister's Prizes for Science.

"Once dispersed, the bacteria are easier to deal with - creating the potential to remove recalcitrant, antimicrobial-tolerant biofilm infections."

University of New South Wales

Nanotechnology World Association

Scientists manipulate consciousness in rats

NIH-funded study may guide deep brain stimulation therapies used for neurological disorders

Scientists showed that they could alter brain activity of rats and either wake them up or put them in an unconscious state by changing the firing rates of neurons in the central thalamus, a region known to regulate arousal. The study, published ineLIFE, was partially funded by the National Institutes of Health.

"Our results suggest the central thalamus works like a radio dial that tunes the brain to different states of activity and arousal," said Jin Hyung Lee, Ph.D., assistant professor of neurology, neurosurgery and bioengineering at Stanford University, and a senior author of the study.

Located deep inside the brain the thalamus acts as a relay station sending neural signals from the body to the cortex. Damage to neurons in the central part of the thalamus may lead to problems with sleep, attention, and memory. Previous studies suggested that stimulation of thalamic neurons may awaken patients who have suffered a traumatic brain injury from minimally conscious states.

Dr. Lee's team flashed laser pulses onto light sensitive central thalamic neurons of sleeping rats, which caused the cells to fire. High frequency stimulation of 40 or 100 pulses per second woke the rats. In contrast, low frequency stimulation of 10 pulses per second sent the rats into a state reminiscent of absence seizures that caused them to stiffen and stare before returning to sleep.

"This study takes a big step towards understanding the brain circuitry that controls sleep and arousal," Yejun (Janet) He, Ph.D., program director at NIH's National Institute of Neurological Disorders and Stroke (NINDS).

When the scientists used functional magnetic resonance imaging (fMRI) to scan brain activity, they saw that high and low frequency stimulation put the rats in completely different states of activity.

Cortical brain areas where activity was elevated during high frequency stimulation became inhibited with low frequency stimulation. Electrical recordings confirmed the results. Neurons in the somatosensory cortex fired more during high frequency stimulation of the central thalamus and less during low frequency stimulation.

"Dr. Lee's innovative work demonstrates the power of using imaging technologies to study the brain at work," said Guoying Liu, Ph.D., a program director at the NIH's National Institute of Biomedical Imaging and Bioengineering (NIBIB).

How can changing the firing rate of the same neurons in one region lead to different effects on the rest of the brain?

Further experiments suggested the different effects may be due to a unique firing pattern by inhibitory neurons in a neighboring brain region, the zona incerta, during low frequency stimulation. Cells in this brain region have been shown to send inhibitory signals to cells in the sensory cortex.

Electrical recordings showed that during low frequency stimulation of the central thalamus, zona incerta neurons fired in a spindle pattern that often occurs during sleep. In contrast, sleep spindles did not occur during high frequency stimulation. Moreover, when the scientists blocked the firing of the zona incerta neurons during low frequency stimulation of the central thalamus, the average activity of sensory cortex cells increased.

Although deep brain stimulation of the thalamus has shown promise as a treatment for traumatic brain injury, patients who have decreased levels of consciousness show slow progress through these treatments.

"We showed how the circuits of the brain can regulate arousal states," said Dr. Lee. "We hope to use this knowledge to develop better treatments for brain injuries and other neurological disorders."

National Institute of Neurological Disorders and Stroke

Nanotechnology World Association