Automating DNA origami opens many new uses

Automating DNA origami opens door to many new uses. Like 3-D printing did for larger objects, method makes it easy to build nanoparticles out of DNA.

Scientia — Researchers can build complex, nanometer-scale structures of almost any shape and form, using strands of DNA. But these particles must be designed by hand, in a complex and laborious process.

3D surface- versus DNA-based renderings of diverse DNA nanoparticles designed autonomously by the algorithm DAEDALUS.
image by Digizyme

This has limited the technique, known as DNA origami, to just a small group of experts in the field.

Now a team of researchers at MIT and elsewhere has developed an algorithm that can build these DNA nanoparticles automatically.

In this way the algorithm, which is reported together with a novel synthesis approach in the journal Science this week, could allow the technique to be used to develop nanoparticles for a much broader range of applications, including scaffolds for vaccines, carriers for gene editing tools, and in archival memory storage.

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Unlike traditional DNA origami, in which the structure is built up manually by hand, the algorithm starts with a simple, 3-D geometric representation of the final shape of the object, and then decides how it should be assembled from DNA, according to Mark Bathe, an associate professor of biological engineering at MIT, who led the research.

“The paper turns the problem around from one in which an expert designs the DNA needed to synthesize the object, to one in which the object itself is the starting point, with the DNA sequences that are needed automatically defined by the algorithm,” Bathe says. “Our hope is that this automation significantly broadens participation of others in the use of this powerful molecular design paradigm.”

The algorithm first represents the object as a perfectly smooth, continuous outline of its surface. It then breaks the surface up into a series of polygonal shapes.

Next, it routes a long, single strand of DNA, called the scaffold, which acts like a piece of thread, throughout the entire structure to hold it together.

A new algorithm for DNA origami starts with a simple, 3-D geometric representation of the final shape of the object, and then decides how it should be assembled from DNA.
Image courtesy of the researchers.

The algorithm weaves the scaffold in one fast and efficient step, which can be used for any shape of 3-D object, Bathe says.

“That [step] is a powerful part of the algorithm, because it does not require any manual or human interface, and it is guaranteed to work for any 3-D object very efficiently,” he says.

The algorithm, which is known as DAEDALUS (DNA Origami Sequence Design Algorithm for User-defined Structures) after the Greek craftsman and artist who designed labyrinths that resemble origami’s complex scaffold structures, can build any type of 3-D shape, provided it has a closed surface. This can include shapes with one or more holes, such as a torus.

In contrast, a previous algorithm, published last year in the journal Nature, is only capable of designing and building the surfaces of spherical objects, and even then still requires manual intervention.

Most previous work on DNA origami has explored 2-D and 3-D structures whose design required some steps to be done by hand, says Paul Rothemund, a research professor at Caltech, who was not involved in the paper.

“The current work provides a complete pipeline, starting from a 3-D form, and arriving at a DNA design and a corresponding predicted atomic model which can be compared quantitatively with experiments,” he says.

The team’s strategy in designing and synthesizing the DNA nanoparticles was also validated using 3-D cryo-electron microscopy reconstructions by Bathe’s collaborator, Wah Chiu at Baylor College of Medicine.

The researchers are now investigating a number of applications for the DNA nanoparticles built by the DAEDALUS algorithm. One such application is a scaffold for viral peptides and proteins for use as vaccines.

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The surface of the nanoparticles could be designed with any combination of peptides and proteins, located at any desired location on the structure, in order to mimic the way in which a virus appears to the body’s immune system.

The researchers demonstrated that the DNA nanoparticles are stable for more than six hours in serum, and are now attempting to increase their stability further.

The nanoparticles could also be used to encapsulate the CRISPR-Cas9 gene editing tool. The CRISPR-Cas9 tool has enormous potential in therapeutics, thanks to its ability to edit targeted genes. However, there is a significant need to develop techniques to package the tool and deliver it to specific cells within the body, Bathe says.

This is currently done using viruses, but these are limited in the size of package they can carry, restricting their use. The DNA nanoparticles, in contrast, are capable of carrying much larger gene packages and can easily be equipped with molecules that help target the right cells or tissue.

The team is also investigating the use of the nanoparticles as DNA memory blocks. Previous research has shown that information can be stored in DNA, in a similar way to the 0s and 1s used to store data digitally. The information to be stored is “written” using DNA synthesis and can then be read back using DNA sequencing technology.

“The current work provides a complete pipeline, starting from a 3-D form, and arriving at a DNA design and a corresponding predicted atomic model which can be compared quantitatively with experiments,” says MIT’s Mark Bathe.
Image courtesy of the researchers.

Using the DNA nanoparticles would allow this information to be stored in a structured and protected way, with each particle akin to a page or chapter of a book. Recalling a particular chapter or book would then be as simple as reading that nanoparticle’s identity, somewhat like using library index cards, Bathe says.

The most exciting aspect of the work, however, is that it should significantly broaden participation in the application of this technology, Bathe says, much like 3-D printing has done for complex 3-D geometric models at the macroscopic scale.

Bathe’s co-authors on the paper are Rémi Veneziano, a postdoc in the Department of Biological Engineering; Sakul Ratanalert, a graduate student in the departments of Biological Engineering and Chemical Engineering; and others from Baylor College of Medicine and Arizona State University.

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– Credit and Resource –

MIT | Helen Knight

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Automating DNA origami opens many new uses

Programming language for living cells

A programming language for living cells. New programming language lets researchers design novel biological circuits.

Scientia — MIT biological engineers have created a programming language that allows them to rapidly design complex, DNA-encoded circuits that give new functions to living cells.

Using this language, anyone can write a program for the function they want, such as detecting and responding to certain environmental conditions. They can then generate a DNA sequence that will achieve it.

“It is literally a programming language for bacteria,” says Christopher Voigt, an MIT professor of biological engineering. “You use a text-based language, just like you’re programming a computer. Then you take that text and you compile it and it turns it into a DNA sequence that you put into the cell, and the circuit runs inside the cell.”

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Voigt and colleagues at Boston University and the National Institute of Standards and Technology have used this language, which they describe in the April 1 issue of Science, to build circuits that can detect up to three inputs and respond in different ways. Future applications for this kind of programming include designing bacterial cells that can produce a cancer drug when they detect a tumor, or creating yeast cells that can halt their own fermentation process if too many toxic byproducts build up.

The researchers plan to make the user design interface available on the Web.

No experience needed

Over the past 15 years, biologists and engineers have designed many genetic parts, such as sensors, memory switches, and biological clocks, that can be combined to modify existing cell functions and add new ones.

However, designing each circuit is a laborious process that requires great expertise and often a lot of trial and error. “You have to have this really intimate knowledge of how those pieces are going to work and how they’re going to come together,” Voigt says.

Users of the new programming language, however, need no special knowledge of genetic engineering.

“You could be completely naive as to how any of it works. That’s what’s really different about this,” Voigt says. “You could be a student in high school and go onto the Web-based server and type out the program you want, and it spits back the DNA sequence.”

The language is based on Verilog, which is commonly used to program computer chips. To create a version of the language that would work for cells, the researchers designed computing elements such as logic gates and sensors that can be encoded in a bacterial cell’s DNA. The sensors can detect different compounds, such as oxygen or glucose, as well as light, temperature, acidity, and other environmental conditions. Users can also add their own sensors. “It’s very customizable,” Voigt says.

The biggest challenge, he says, was designing the 14 logic gates used in the circuits so that they wouldn’t interfere with each other once placed in the complex environment of a living cell.

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In the current version of the programming language, these genetic parts are optimized for E. coli, but the researchers are working on expanding the language for other strains of bacteria, including Bacteroides, commonly found in the human gut, and Pseudomonas, which often lives in plant roots, as well as the yeast Saccharomyces cerevisiae. This would allow users to write a single program and then compile it for different organisms to get the right DNA sequence for each one.

Biological circuits

Using this language, the researchers programmed 60 circuits with different functions, and 45 of them worked correctly the first time they were tested. Many of the circuits were designed to measure one or more environmental conditions, such as oxygen level or glucose concentration, and respond accordingly. Another circuit was designed to rank three different inputs and then respond based on the priority of each one.

One of the new circuits is the largest biological circuit ever built, containing seven logic gates and about 12,000 base pairs of DNA.

Another advantage of this technique is its speed. Until now, “it would take years to build these types of circuits. Now you just hit the button and immediately get a DNA sequence to test,” Voigt says.

His team plans to work on several different applications using this approach: bacteria that can be swallowed to aid in digestion of lactose; bacteria that can live on plant roots and produce insecticide if they sense the plant is under attack; and yeast that can be engineered to shut off when they are producing too many toxic byproducts in a fermentation reactor.

The lead author of the Science paper is MIT graduate student Alec Nielsen. Other authors are former MIT postdoc Bryan Der, MIT postdoc Jonghyeon Shin, Boston University graduate student Prashant Vaidyanathan, Boston University associate professor Douglas Densmore, and National Institute of Standards and Technology researchers Vanya Paralanov, Elizabeth Strychalski, and David Ross.

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– Credit and Resource –

Anne Trafton | MIT News Office

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Programming language for living cells

Process to Accelerate Protein Evolution

Stanford engineers invent process to accelerate protein evolution

A new tool enables researchers to test millions of mutated proteins in a matter of hours or days, speeding the search for new medicines, industrial enzymes and biosensors.

All living things require proteins, members of a vast family of molecules that nature “makes to order” according to the blueprints in DNA.

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Through the natural process of evolution, DNA mutations generate new or more effective proteins. Humans have found so many alternative uses for these molecules – as foods, industrial enzymes, anti-cancer drugs – that scientists are eager to better understand how to engineer protein variants designed for specific uses.

An overview of the directed evolution process using the new Stanford technique: preparing protein libraries, screening them, extracting desired cells, and then inferring their DNA sequence. (Click image to enlarge.)

Now Stanford engineers have invented a technique to dramatically accelerate protein evolution for this purpose. This technology, described in Nature Chemical Biology, allows researchers to test millions of variants of a given protein, choose the best for some task and determine the DNA sequence that creates this variant.

“Evolution, the survival of the fittest, takes place over a span of thousands of years, but we can now direct proteins to evolve in hours or days,” said Jennifer Cochran, an associate professor of bioengineering who co-authored the paper with Thomas Baer, executive director of the Stanford Photonics Research Center.

“This is a practical, versatile system with broad applications that researchers will find easy to use,” Baer said.

By combining Cochran’s protein engineering know-how with Baer’s expertise in laser-based instrumentation, the team created a tool that can test millions of protein variants in a matter of hours.

“The demonstrations are impressive and I look forward to seeing this technology more widely adopted,” said Frances Arnold, a professor of chemical engineering at Caltech who was not affiliated with the study.

Making a million mutants

The researchers call their tool µSCALE, or Single Cell Analysis and Laser Extraction.

The “µ” stands for the microcapillary glass slide that holds the protein samples. The slide is roughly the size and thickness of a penny, yet in that space a million capillary tubes are arrayed like straws, open on the top and bottom.

The microcapillary glass slide, roughly the size and thickness of a penny, holds the protein samples.

The power of µSCALE is how it enables researchers to build upon current biochemical techniques to run a million protein experiments simultaneously, then extract and further analyze the most promising results.

The researchers first employ a process termed “mutagenesis” to create random variations in a specific gene. These mutations are inserted into batches of yeast or bacterial cells, which express the altered gene and produce millions of random protein variants.

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A µSCALE user mixes millions of tiny opaque glass beads into a sample containing millions of yeast or bacteria and spreads the mixture on a microcapillary slide. Tiny amounts of fluid trickle into each tube, carrying individual cells. Surface tension traps the liquid and the cell in each capillary.

The slide bearing these million yeast or bacteria, and the protein variants they produce, is inserted into the µSCALE device. A software-controlled microscope peers into each capillary and takes images of the biochemical reaction occurring therein.

Once a µSCALE user identifies a capillary of interest, the researcher can direct the laser to extract the contents of that tube without disrupting its neighbors, using an ingenious method devised by Baer.

“The beads are what enable extraction,” Baer said. “The laser supplies energy to move the beads, which breaks the surface tension and releases the sample from the capillary.”

Thus µSCALE empties the contents of a single capillary onto a collector plate, where the DNA of the isolated cell can be sequenced and the gene variant responsible for the protein of interest can be identified.

“One of the unique features of µSCALE is that it allows researchers to rapidly isolate a single desired cell from hundreds of thousands of other cells,” said Bob Chen, a doctoral student in Cochran’s lab who wrote the software to examine and detect signs of interesting protein activity within the test tubes.

Promising variants can be collected and reprocessed through µSCALE to further evolve and optimize the protein.

“This is an exciting new tool to answer important questions about proteins,” Cochran said, likening µSCALE to the way that high-throughput tools for gene analysis have allowed researchers to unlock key features of biology underlying human disease.

Genesis and proofs

The project began five years ago when Baer and collaborator Ivan Dimov developed the first instrument. They showed how to identify cell types in a microcapillary array and extract a single capillary’s contents using glass beads and a focused laser.

About three years ago, Cochran and Baer joined forces to develop µSCALE for protein engineering, and the team devised three experiments to showcase µSCALE’s utility and flexibility.

In one experiment, researchers sifted through a protein library produced in yeast cells to select antibodies that bound most tightly to a cancer target. Antibodies with a high target-binding affinity are known to be effective against cancer.

In a second example, they engineered a bright orange fluorescent protein biosensor. Using µSCALE, they did this almost 10 times faster than previous methods. Such biosensors are often used as tags in a wide variety of biology experiments.

A third experiment, carried out with Stanford biochemistry Professor Daniel Herschlag, used µSCALE to improve upon a model enzyme.

“This system will allow us to explore the evolutionary and functional relationships between enzymes, guiding the engineering of new enzymes that can carry out novel beneficial reactions,” Herschlag said.

The Stanford team included graduate students Sungwon Lim and Arvind Kannan, postdoctoral scholar Spencer Alford and researcher Fanny Sunden.

Support for this work included a Wallace H. Coulter Translational Partnership Award, which made this pioneering interdisciplinary research possible.

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– Credit and Resource –

Written by: Ramin Skibba

Provided by: Standford University

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Process to Accelerate Protein Evolution

Drug-resistance Mechanism in Tumor Cells

Biologists unravel drug-resistance mechanism in tumor cells – Targeting the RNA-binding protein that promotes resistance could lead to better cancer therapies.

P53, which helps healthy cells prevent genetic mutations, is missing from about half of all tumors. Researchers have found that a backup system takes over when p53 is disabled and encourages cancer cells to continue dividing. In the background of this illustration are crystal structures of p53 DNA-binding domains.
Image: Jose-Luis Olivares/MIT (p53 illustration by Richard Wheeler/Wikimedia Commons)

Scientia — About half of all tumors are missing a gene called p53, which helps healthy cells prevent genetic mutations. Many of these tumors develop resistance to chemotherapy drugs that kill cells by damaging their DNA.

MIT cancer biologists have now discovered how this happens: A backup system that takes over when p53 is disabled encourages cancer cells to continue dividing even when they have suffered extensive DNA damage. The researchers also discovered that an RNA-binding protein called hnRNPA0 is a key player in this pathway.

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“I would argue that this particular RNA-binding protein is really what makes tumor cells resistant to being killed by chemotherapy when p53 is not around,” says Michael Yaffe, the David H. Koch Professor in Science, a member of the Koch Institute for Integrative Cancer Research, and the senior author of the study, which appears in the Oct. 22 issue of Cancer Cell.

The findings suggest that shutting off this backup system could make p53-deficient tumors much more susceptible to chemotherapy. It may also be possible to predict which patients are most likely to benefit from chemotherapy and which will not, by measuring how active this system is in patients’ tumors.

Rewired for resistance

In healthy cells, p53 oversees the cell division process, halting division if necessary to repair damaged DNA. If the damage is too great, p53 induces the cell to undergo programmed cell death.

In many cancer cells, if p53 is lost, cells undergo a rewiring process in which a backup system, known as the MK2 pathway, takes over part of p53’s function. The MK2 pathway allows cells to repair DNA damage and continue dividing, but does not force cells to undergo cell suicide if the damage is too great. This allows cancer cells to continue growing unchecked after chemotherapy treatment.

“It only rescues the bad parts of p53’s function, but it doesn’t rescue the part of p53’s function that you would want, which is killing the tumor cells,” says Yaffe, who first discovered this backup system in 2013.

In the new study, the researchers delved further into the pathway and found that the MK2 protein exerts control by activating the hnRNPA0 RNA-binding protein.

RNA-binding proteins are proteins that bind to RNA and help control many aspects of gene expression. For example, some RNA-binding proteins bind to messenger RNA (mRNA), which carries genetic information copied from DNA. This binding stabilizes the mRNA and helps it stick around longer so the protein it codes for will be produced in larger quantities.

“RNA-binding proteins, as a class, are becoming more appreciated as something that’s important for response to cancer therapy. But the mechanistic details of how those function at the molecular level are not known at all, apart from this one,” says Ian Cannell, a research scientist at the Koch Institute and the lead author of the Cancer Cell paper.

In this paper, Cannell found that hnRNPA0 takes charge at two different checkpoints in the cell division process. In healthy cells, these checkpoints allow the cell to pause to repair genetic abnormalities that may have been introduced during the copying of chromosomes.

One of these checkpoints, known as G2/M, is controlled by a protein called Gadd45, which is normally activated by p53. In lung cancer cells without p53, hnRNPA0 stabilizes mRNA coding for Gadd45. At another checkpoint called G1/S, p53 normally turns on a protein called p21. When p53 is missing, hnRNPA0 stabilizes mRNA for a protein called p27, a backup to p21. Together, Gadd45 and p27 help cancer cells to pause the cell cycle and repair DNA so they can continue dividing.

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Personalized medicine

The researchers also found that measuring the levels of mRNA for Gadd45 and p27 could help predict patients’ response to chemotherapy. In a clinical trial of patients with stage 2 lung tumors, they found that patients who responded best had low levels of both of those mRNAs. Those with high levels did not benefit from chemotherapy.

“You could measure the RNAs that this pathway controls, in patient samples, and use that as a surrogate for the presence or absence of this pathway,” Yaffe says. “In this trial, it was very good at predicting which patients responded to chemotherapy and which patients didn’t.”

“The most exciting thing about this study is that it not only fills in gaps in our understanding of how p53-deficient lung cancer cells become resistant to chemotherapy, it also identifies actionable events to target and could help us to identify which patients will respond best to cisplatin, which is a very toxic and harsh drug,” says Daniel Durocher, a senior investigator at the Samuel Lunenfeld Research Institute of Mount Sinai Hospital in Toronto, who was not part of the research team.

The MK2 pathway could also be a good target for new drugs that could make tumors more susceptible to DNA-damaging chemotherapy drugs. Yaffe’s lab is now testing potential drugs in mice, including nanoparticle-based sponges that would soak up all of the RNA binding protein so it could no longer promote cell survival.

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– Credit and Resource –

Anne Trafton | MIT News Office

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Drug-resistance Mechanism in Tumor Cells

New test detects all viruses

A new test developed at Washington University School of Medicine in St. Louis can detect virtually any virus that infects people and animals, including the Ebola virus (above). Credit: National Institute of Allergy and Infectious Diseases

Scientia — A new test detects virtually any virus that infects people and animals, according to research at Washington University School of Medicine in St. Louis, where the technology was developed.

Many thousands of viruses are known to cause illness in people and animals, and making a diagnosis can be an exhaustive exercise, at times requiring a battery of different tests. That’s because current tests aren’t sensitive enough to detect low levels of viral bugs or are limited to detecting only those viruses suspected of being responsible for a patient’s illness.

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“With this test, you don’t have to know what you’re looking for,” said the study’s senior author, Gregory Storch, MD, the Ruth L. Siteman Professor of Pediatrics. “It casts a broad net and can efficiently detect viruses that are present at very low levels. We think the test will be especially useful in situations where a diagnosis remains elusive after standard testing or in situations in which the cause of a disease outbreak is unknown.”

Results published online in September in the journal Genome Research demonstrate that in patient samples the new test – called ViroCap – can detect viruses not found by standard testing based on genome sequencing. The new test could be used to detect outbreaks of deadly viruses such as Ebola, Marburg and severe acute respiratory syndrome (SARS), as well as more routine viruses, including rotavirus and norovirus, both of which cause severe gastrointestinal infections.

The test sequences and detects viruses in patient samples and is just as sensitive as the gold-standard polymerase chain reaction (PCR) assays, which are used widely in clinical laboratories. However, even the most expansive PCR assays can only screen for up to about 20 similar viruses at the same time.

The Washington University researchers are making the technology they developed publicly available to scientists and clinicians worldwide, for the benefit of patients and research.

The researchers evaluated the new test in two sets of biological samples – for example, from blood, stool and nasal secretions – from patients at St. Louis Children’s Hospital. In the first, standard testing that relied on genome sequencing had detected viruses in 10 of 14 patients. But the new test found viruses in the four children that earlier testing had missed. Standard testing failed to detect common, everyday viruses: influenza B, a cause of seasonal flu; parechovirus, a mild gastrointestinal and respiratory virus; herpes virus 1, responsible for cold sores in the mouth; and varicella-zoster virus, which causes chickenpox.

In a second group of children with unexplained fevers, standard testing had detected 11 viruses in the eight children evaluated. But the new test found another seven, including a respiratory virus called human adenovirus B type 3A, which usually is harmless but can cause severe infections in some patients.

In all, the number of viruses detected in the two patient groups jumped to 32 from 21, a 52 percent increase.

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“The test is so sensitive that it also detects variant strains of viruses that are closely related genetically,” said corresponding author Todd Wylie, an instructor of pediatrics. “Slight genetic variations among viruses often can’t be distinguished by currently available tests and complicate physicians’ ability to detect all variants with one test.”

In addition, because the test includes detailed genetic information about various strains of particular viruses, subtypes can be identified easily. For example, the study showed that while standard testing identified a virus as influenza A, which causes seasonal flu, the new test indicated that the virus was a particularly harsh subtype called H3N2.

Last flu season, H3N2 contributed to some 36,000 deaths in the United States. And in some patients – particularly young children, older adults and people with weakened immune systems – knowing that the H3N2 strain is present may alter treatment.

To develop the test, the researchers targeted unique stretches of DNA or RNA from every known group of viruses that infects humans and animals. In all, the research team included 2 million unique stretches of genetic material from viruses in the test. These stretches of material are used as probes to pluck out viruses in patient samples that are a genetic match. The matched viral material then is analyzed using high-throughput genetic sequencing. As completely novel viruses are discovered, their genetic material could easily be added to the test, Storch said.

The researchers plan to conduct additional research to validate the accuracy of the test, so it could be several years before it is clinically available.

“It also may be possible to modify the test so that it could be used to detect pathogens other than viruses, including bacteria, fungi and other microbes, as well as genes that would indicate the pathogen is resistant to treatment with antibiotics or other drugs,” said co-author Kristine Wylie, PhD, assistant professor of pediatrics.

In the meantime, the technology can be used by scientists to study viruses in a research setting. Kristine Wylie investigates the viruses that set up residence in and on the human body, collectively known as the virome. The new test will provide a way to capture the full breadth and depth of such viruses, and deepen understanding of how they play a role in keeping the body healthy.

Pictures of Viruses under an Electron Microscope

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– Credit and Resource –

More information: Wylie TN, Wylie KM, Herter BN and Storch GA. Enhanced virome sequencing using targeted sequence capture. Genome Research, online Sept. 22, 2015.

Journal reference: Genome Research

Provided by: Washington University School of Medicine

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New test detects all viruses

Targeting DNA

Protein-based sensor could detect viral infection or kill cancer cells.

Scientia — MIT biological engineers have developed a modular system of proteins that can detect a particular DNA sequence in a cell and then trigger a specific response, such as cell death.

This system can be customized to detect any DNA sequence in a mammalian cell and then trigger a desired response, including killing cancer cells or cells infected with a virus, the researchers say.

“There is a range of applications for which this could be important,” says James Collins, the Termeer Professor of Medical Engineering and Science in MIT’s Department of Biological Engineering and Institute of Medical Engineering and Science (IMES). “This allows you to readily design constructs that enable a programmed cell to both detect DNA and act on that detection, with a report system and/or a respond system.”

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Collins is the senior author of a Sept. 21 Nature Methods paper describing the technology, which is based on a type of DNA-binding proteins known as zinc fingers. These proteins can be designed to recognize any DNA sequence.

“The technologies are out there to engineer proteins to bind to virtually any DNA sequence that you want,” says Shimyn Slomovic, an IMES postdoc and the paper’s lead author. “This is used in many ways, but not so much for detection. We felt that there was a lot of potential in harnessing this designable DNA-binding technology for detection.”

Sense and respond

To create their new system, the researchers needed to link zinc fingers’ DNA-binding capability with a consequence — either turning on a fluorescent protein to reveal that the target DNA is present or generating another type of action inside the cell.

The researchers achieved this by exploiting a type of protein known as an “intein” — a short protein that can be inserted into a larger protein, splitting it into two pieces. The split protein pieces, known as “exteins,” only become functional once the intein removes itself while rejoining the two halves.

Collins and Slomovic decided to divide an intein in two and then attach each portion to a split extein half and a zinc finger protein. The zinc finger proteins are engineered to recognize adjacent DNA sequences within the targeted gene, so if they both find their sequences, the inteins line up and are then cut out, allowing the extein halves to rejoin and form a functional protein. The extein protein is a transcription factor designed to turn on any gene the researchers want.

In this paper, they linked green fluorescent protein (GFP) production to the zinc fingers’ recognition of a DNA sequence from an adenovirus, so that any cell infected with this virus would glow green.

This approach could be used not only to reveal infected cells, but also to kill them. To achieve this, the researchers could program the system to produce proteins that alert immune cells to fight the infection, instead of GFP.

“Since this is modular, you can potentially evoke any response that you want,” Slomovic says. “You could program the cell to kill itself, or to secrete proteins that would allow the immune system to identify it as an enemy cell so the immune system would take care of it.”

Martin Fussenegger, a professor of biotechnology and bioengineering at the Swiss Federal Institute of Technology in Zurich, described this experiment as an “elegant proof of concept” that could lead to greatly improved treatments for viral infection.

“Sentinel designer cells engineered with the DNA sense-and-response system may one day be able to sense and eliminate viruses in our body. This would represent a quantum leap in antiviral therapy,” says Fussenegger, who was not involved in the study.

The MIT researchers also deployed this system to kill cells by linking detection of the DNA target to production of an enzyme called NTR. This enzyme activates a harmless drug precursor called CB 1954, which the researchers added to the petri dish where the cells were growing. When activated by NTR, CB 1954 kills the cells.

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Future versions of the system could be designed to bind to DNA sequences found in cancerous genes and then produce transcription factors that would activate the cells’ own programmed cell death pathways.

Research tool

The researchers are now adapting this system to detect latent HIV proviruses, which remain dormant in some infected cells even after treatment. Learning more about such viruses could help scientists find ways to permanently eliminate them.

“Latent HIV provirus is pretty much the final barrier to curing AIDS, which currently is incurable simply because the provirus sequence is there, dormant, and there aren’t any ways to eradicate it,” Slomovic says.

While treating diseases using this system is likely many years away, it could be used much sooner as a research tool, Collins says. For example, scientists could use it to test whether genetic material has been successfully delivered to cells that scientists are trying to genetically alter. Cells that did not receive the new gene could be induced to undergo cell death, creating a pure population of the desired cells.

It could also be used to study chromosomal inversions and transpositions that occur in cancer cells, or to study the 3-D structure of normal chromosomes by testing whether two genes located far from each other on a chromosome fold in such a way that they end up next to each other, the researchers say.

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– Credit and Resource –

Anne Trafton | MIT News Office

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Targeting DNA