Researchers have discovered the details of how cells repair breaks in both strands of DNA, a potentially devastating kind of DNA damage.
When chromosomes experience double-strand breaks due to oxidation, ionizing radiation, replication errors and certain metabolic products, cells utilize their genetically similar chromosomes to patch the gaps via a mechanism that involves both ends of the broken molecules. To repair a broken chromosome that lost one end, a unique configuration of the DNA replication machinery is deployed as a desperation strategy to allow cells to survive, the researchers discovered.
The collaborative work of graduate students working under Anna Malkova, associate professor of biology at Indiana University-Purdue University Indianapolis (IUPUI) and Kirill Lobachev, associate professor of biology at the Georgia Institute of Technology, was critical in the advancement of the project. The group’s research was scheduled to be published Sept. 11 in the online edition of the journal Nature, with two graduate students, Sreejith Ramakrishnan of IUPUI, and Natalie Saini of Georgia Tech, as first authors. Other collaborators include James Haber of Brandeis University and Grzegorz Ira of the Baylor College of Medicine.
“Previously we have shown that the rate of mutations introduced by break-induced replication is 1,000 times higher as compared to the normal way that DNA is made naturally, but we never understood why,” Malkova said.
Lobachev’s lab used cutting-edge analysis techniques and equipment available at only a handful of labs around the world. This allowed the researchers to see inside yeast cells and freeze the break-induced DNA repair process at different times. They found that this mode of DNA repair doesn’t rely on the traditional replication fork — a Y-shaped region of a replicating DNA molecule — but instead uses a bubble-like structure to synthesize long stretches of missing DNA. This bubble structure copies DNA in a manner not seen before in eukaryotic cells.
Traditional DNA synthesis, performed during the S-phase of the cell cycle, is done in semi-conservative manner as shown by Matthew Meselson and Franklin Stahl in 1958 shortly after the discovery of the DNA structure. They found that two new double helices of DNA are produced from a single DNA double helix, with each new double helix containing one original strand of DNA and one new strand.
“We demonstrated that break-induced replication differs from S-phase DNA replication as it is carried out by a migrating bubble instead of a normal replication fork and leads to conservative DNA synthesis promoting highly increased mutagenesis,” Malkova said.
This desperation replication triggers “bursts of genetic instability” and could be a contributing factor in tumor formation.
“From the point of view of the cell, the whole idea is to survive, and this is a way for them to survive a potentially lethal event, but it comes at a cost,” Lobachev said. “Potentially, it’s a textbook discovery.”
During break-induced replication, one broken end of DNA is paired with an identical DNA sequence on its partner chromosome. Replication that proceeds in an unusual bubble-like mode then copies hundreds of kilobases of DNA from the donor DNA through the telomere at the ends of chromosomes.
“Surprisingly, this is a way of synthesizing DNA in a very robust manner,” Saini said. “The synthesis can take place and cover the whole arm of the chromosome, so it’s not just some short patches of synthesis.”
The bubble-like mode of DNA replication can operate in non-dividing cells, which is the state of most of the body’s cells, making this kind of replication a potential route for cancer formation.
“Importantly, the break-induced replication bubble has a long tail of single-stranded DNA, which promotes mutations,” Ramakrishnan said.
The single-stranded tail might be responsible for the high mutation-rate because it can accumulate mutations by escaping the other repair mechanisms that quickly detect and correct errors in DNA synthesis.
“When it comes to cancer, other diseases and even evolution, what seems to be happening are bursts of instability, and the mechanisms promoting such bursts were unclear,” Malkova said.
The molecular mechanism of break-induced replication unraveled by the new study provides one explanation for the generation of mutations.
This research is supported by the National Institutes of Health under awards RO1GM082950, RO1GM084242, RO3ES016434, GM76020, and by the National Science Foundation under award MCB-0818122. Any conclusions or opinions are those of the authors and do not necessarily represent the official views of the NIH or NSF.
CITATION: N. Saini, et al., “Migrating bubble during break-induced replication drives conservative DNA synthesis,” (Nature, 2013). http://dx.doi.org/10.1038/nature12584
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The National Science Foundation has awarded a 5 year grant of approximately $2.0 million to fund a collaborative group of scientists: Mark Young (PI, Montana State), Joshua Weitz (Co-PI, Georgia Tech), and Rachel Whitaker (Co-PI, UIUC) to study the role of viruses in shaping genetic, taxonomic and functional diversity.
The team will investigate a new hypothesis about how viruses may control the structure and function of microbial communities. The traditional view of viruses is that they negatively impact the fitness of infected hosts. In other words, they are viewed strictly as pathogens, in which the host tries to eliminate the virus. This project will explore an alternative hypothesis: that chronic viral infections contribute positively to host fitness, increasing the success of the virus-host pair by protecting their hosts from infection by even more pathogenic viruses.
Two proposals by Georgia Tech researchers, Dr. Frank Stewart (Assistant Professor, School of Biology) and Dr. Kostas Konstantinidis (Carlton S. Wilder Assistant Professor, Civil and Environmental Engineering; joint appointment in Biology; http://enve-omics.gatech.edu), have been selected for the Department of Energy's 2014 Community Science Program. The CSP provides high-throughput DNA sequencing resources to support genomics research of relevance to urgent energy and environmental challenges. Dr. Stewart's project seeks to understand how oxygen loss, such as that caused by agricultural runoff, affects microbial pathways of carbon and energy flow in marine ecosystems. Dr. Konstantinidis' project will explore how microbes survive in the extreme environment of the upper troposphere. This project represents a "bold new direction" for the DOE CSP program and may contribute insight into how microbes affect cloud formation and the Earth's water cycle. Additional details about 2014 CSP projects can be found at http://www.jgi.doe.gov/News/news_13_10_28.html
Funding for research is a highly competitive endeavor under the best of circumstances. For Georgia Tech doctoral student Troy Alexander, a new avenue for funding has opened.
Alexander works as a researcher in School of Biology Professor Julia Kubanek’s group. His latest project seeks to accelerate the discovery of new medicines for the treatment of cancer and infectious diseases by studying Fijian red algae.
To help raise funds to support this research, Alexander and Kubanek posted the project on Georgia Tech Starter, a university-based, peer-reviewed crowdfunding platform for faculty-sponsored scientific research projects.
Alexander established a fundraising goal of $9,450 to fast-track the discovery of these new medicines through a combination of biomedical screening, nuclear magnetic resonance spectroscopy, and multivariate statistical analysis of his library of marine chemical compounds from the Fijian organisms. By identifying unknown molecules, the research will prioritize exploration of previously unseen structures that can exhibit strong potency toward microbial and human cancer cell lines.
“These molecules previously unknown to science will be carried forward for purification, structure determination, and development as treatments for disease,” Alexander said.
Georgia Tech Starter is a perfect venue for making human medicine research advances more accessible to a general audience, he added.
According to Allison Mercer, an applied physicist at Georgia Tech Research Institute – and the mastermind behind Georgia Tech Starter – using crowdfunding for science means there is a community of people invested in the research, witnessing its benefits and outcomes.
“There is incredible potential for contributors to engage with scientists in a way that hasn’t been done before: A project that is successfully funded converts into a blog through which only project supporters can monitor the progress of the project, ask the scientists questions, and witness world-class research at Tech as it unfolds.”
But, before taking a step to gain the world’s attention through Tech Starter, researchers need to consider several things, Alexander said: One is the amount of funding to request.
“The amount we seek to raise should be large enough to make an impact on our research, but it shouldn’t be so large to sound insurmountable to potential donors,” he said.
“Another item to consider is how easily one can make their research appeal to a broader audience,” he said. “Do you have any tangible goals that can be achieved within the first year as proof of progress to donors? Are you willing to commit to engaging with the audience – from making the video to maintaining social media contact? These are huge commitments, and they need to be managed without becoming a drain on research time.”
Lastly, Alexander advises that the goals of the project need to be easy to explain and justify. Short-term goals should be clear to give investors something to anticipate. And, he said, researchers should be prepared for the contingency that the funds might not be raised.
Like other crowdfunding platforms, Georgia Tech Starter operates with an all-or-nothing funding strategy. Only if the project goal amount is reached within a 60-day window, are donors’ credit cards charged. This helps assure supporters that the researchers will only receive the pledged money if they have enough total funds to achieve the stated project goals. Some research efforts are scalable, but many are not.
“If it costs a million dollars to launch a telescope into space, it doesn’t do you any good to get halfway there,” said Mercer.
Students interested in taking advantage of Tech’s crowdfunding site can visit starter.gatech.edu to submit their contact information.
The process for getting a project posted on Georgia Tech Starter includes a comprehensive peer review of the project to ensure that: it is achievable; the requested funding amount is enough to complete the project; and the researchers on the project have the knowledge, skills, and abilities to get the research done right. Through the peer review process, researchers will receive feedback on how to better craft the project’s message for posting.
“We do everything we can to support the project creators so they can be successful,” said Mercer.
Danielle Dixson is a new faculty member in the School of Biology this year, but she’s not new to Georgia Tech. She spent the previous two years as a post-doctoral fellow in Professor Mark Hay’s lab. Before that she received her Ph.D. from James Cook University in Australia and her B.S. from the University of Tampa. One might say she was brought up with biology in her future … the Minnesota Zoo was right behind her back fence as a kid.
Danielle Dixson: The Minnesota zoo has a special kind of school, kind of like a flagship school, it’s called the School of Environmental Studies. It’s actually at the zoo. So you take all your classes your junior and senior year at the zoo, and they incorporate biology into everything that you’re doing. So I got to take marine biology in Minnesota as a junior, because we got to use the aquariums there.
David Terraso: So, is that where your interest in biology began?
Dixson: I’m one of those kids who, when I was five, I said I wanted to be a marine biologist and my parents were like ok. And to anyone who asked, I said, “Oh, I want to be a marine biologist.” They said, “Oh, ok,” thinking I would grow out of it or something. It’s like every little kids dream, but I never changed my mind.
Terraso: Tell us about your research and what you’re looking to do in the next few years.
Dixson: My research in general is how do chemical cues, or smells in the water, give information that cause a behavioral response in fish. So, what smells elicit certain behavioral patterns, and how does that reflect in community dynamics and settlement selection.
A lot of my work looks at larval fish, or juvenile fishes. And when marine fish reproduce they lay eggs, or spawn into the water column, and the larva, or the eggs, go off into the pelagic environment, and they need to come back to the reef. And what I’m trying to figure out is what chemical cues do they use to decide what reef is a good reef and what reef is a bad reef.
Terraso: Tell us about some of your research projects.
Dixson: So a big project of mine in Fiji is looking at the marine protected areas there, looking at how, if you have a protected area there that’s a very pristine, healthy habitat and you have one that’s a very non-pristine protected area (where it’s essentially trashed because people fish in it and are always in it and there’s runoff and a lot of algae and not a lot of fish) looking at how we can get coral back into that non-protected area. And it seems like the chemical cues may be responsible for the coral and fish larva rejecting that area as a habitat, because it’s so different from the healthy area right next door. So that’s been one of the primary focuses of my post-doc, and I’ll continue doing some of that work with Mark Hay as well.
Another thing that I work on is ocean acidification and the effect that that has on behavior and the effect that that has on mostly larval fishes. I’ve started doing some projects on sharks that I’ll continue while at Tech. I’ve been talking to the Georgia Aquarium about collaborating with them and using some of their shark eggs that they get pretty regularly and treating them with different levels of ocean acidification scenarios that are going to be happening in the near future, within the next 100 years, and looking at how that’s affecting the behavioral response of the animals.
Terraso: Looking further into your career, say 30 years from now, what do you want to have accomplished?
Dixson: I guess 30 years out, I’d like to continue in the same role and be able to have provided the marine community with a much better understanding of how chemical cues work in the marine environment and how the sensory system plays a huge roll in the behavior that comes across with fish and coral larvae.
In the recent past, we’ve been thinking these tiny fish larvae, when they were out in the open ocean, that they were just passive particles drifting around with no say in where they were going. And now in a very short time, it’s been shown, mostly through the ability to use genetics in different ways, that they’re actually going to specific places. We don’t know why they’re going to those specific places. We don’t know how they’re able to manage to get to these places, but clearly their behavior is not passive.
They’re overcoming ocean currents. They’re overcoming all of these obstacles that we thought they would not be able to do. And they’re getting to these locations and their behavior is really the only way you can explain it.
In order to do something, you need a motive to do it, and the sensory cues are what provides them information to decide where to go. So I’d really like to get into how different sensory systems interact. So if you get an auditory cue and an olfactory cue and the olfactory cue is a positive stimulus, but the auditory cue might not sound right, which would you follow? What choices will you make?
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Georgia Tech faculty continue to be recognized as among the most respected in their field. Last month, the American Association for the Advancement of Science (AAAS) named four — in biology, computing and engineering — to its 2013 class of fellows
Election as a fellow of AAAS, the world’s largest general scientific society, is an honor bestowed upon members by their peers. Fellows are recognized for meritorious efforts to advance science or its applications.
New fellows include:
- School of Interactive Computing Professor Henrik Christensen, cited “for contributions to applied estimation methods in mapping, robot localization, visual tracking and recognition, as well as national-level leadership of the robotics community.”
- School of Biology Professor Mark Hay, cited “for distinguished contributions in ecology, particularly for developing marine chemical ecology and for elucidating how chemical cues and signals structure populations, communities, and ecosystems.”
- School of Chemical and Biomolecular Engineering Professor Hang Lu, cited “for distinguished contributions to the field of engineering systems for high-throughput quantitative and systems biology, particularly for microfluidics, automation, image-based science, and phenomics.”
- School of Aerospace Engineering Professor Suresh Menon, cited “for distinguished and innovative contributions to the field of multi-scale computational simulation and modeling of turbulent combustion in power and propulsion systems.”
The Parker H. Petit Institute for Bioengineering & Bioscience awarded the 2014 Suddath Symposium Graduate Student Awards to three students for their grand achievements in biological or biochemical research at the molecular or cellular level.
"It was a difficult decision – we had a very strong applicant pool this year," said Nick Hud, Associate Director for the Parker H. Petit Institute for Bioengineering and Bioscience and Professor in the School of Chemistry and Biochemistry.
The first place award was given to Natalie Saini who is pursuing her Ph.D. in Molecular and Cell Biology. Saini’s research is focused on determining the mechanisms underlying erroneous DNA synthesis during double strand break (DSB) repair in eukaryotic cells, an important process implicated in the generation of instability in cancers. Her work has been published in Nature, Biochimie, Molecular Cell and PLoS Genetics..
“I am very honored to receive the prestigious Suddath award,” said Saini. “I am thankful to the reviewers for recognizing my accomplishments and grateful for all of the opportunities and resources that have been provided through my advisor, Kirill Lobachev, as well as through Georgia Tech’s Petit Institute.”
Saini will receive $1,000 and will give a research presentation to the Petit Institute community at the 2014 Suddath Symposium to be held on February 20, 2014 at Georgia Tech. She will also have her name added to the Suddath Award recognition plaque at the Petit Institute.
“Natalie is smart, motivated and hardworking scientist. She has excellent
analytical skills and she is not afraid to try new approaches and techniques,” said her advisor, Kirill Lobachev.
Lauren Austin received the 2nd place award for her research in nanobiotechnology in the laboratory of Mostafa El-Sayed where she is focused on the interactions of plasmonic nanoparticles (NPs) with cancerous cell lines and the exploitation of their unique optical properties to reveal molecular information during important cellular functions (i.e. proliferation, cell cycle progression, and cell death) in real-time.
Anthony Awojoodu, a doctoral student in Biomedical engineering, was recognized for a 3rd place award for his accomplishments in the laboratory of Edward Botchwey, where he has focused his research on therapies to cure, treat and prevent complications of sickle cell disease using sphingolipid signaling and metabolism.
Austin and Awojoodu will also each receive cash awards.
John McDonald, professor in the School of Biology and director of the Integrated Cancer Research Center, has also spent many years as the chief scientific officer for Georgia Tech’s Ovarian Cancer Institute.
Collaboration doesn’t just come easy for him. It is at the very foundation of his research approach when it comes to understanding cancer. McDonald, then, was a natural choice among faculty members who will relocate to the Engineered Biosystems Building (EBB) when it opens in 2015. Campaign Georgia Tech has been instrumental in raising money for the building.
“I’m convinced that the effective treatment of complex diseases like cancer will require an understanding of the interactive relationships that underlie cell function,” McDonald said. “I am excited about the prospect of working with other researchers committed to a ‘systems’ approach to better understand the basis of cancer onset and progression.”
The EBB was conceptualized and designed, and will be constructed, according to one fundamental tenet — that understanding and fighting multifaceted disease requires a new way of doing things; that new insights emerge not from the solitary confines of one laboratory or one discipline but from shared resources, spaces, and expertise.
The collaborative spaces within the facility are decidedly intentional and planned. The five-story, 200,000-square-foot building will house faculty members and other researchers in three research neighborhoods: chemical biology, cell and developmental bioengineering, and systems biology. Within each neighborhood, scientists and engineers from many different disciplines will share lab, office, and communal spaces, making it possible for them to share ideas, perspectives, and resources in an entirely new way.
For many years, McDonald has taken a collaborative approach to cancer research, working with faculty in chemistry and computer science to develop new, highly accurate diagnostic tests for ovarian and prostate cancer, and partnering with biomedical engineers, chemists, and biologists in cell therapies and personalized cancer medicine. Once the EBB is operational, collaboration will drive its every function and use, which will help accelerate the pace of discovery.
“We are not striving to compete with cancer centers like MD Anderson,” explained McDonald. “We are complementing their efforts by developing these unique integrative approaches, and this building will greatly enhance our ability to do that.”
For more about Campaign Georgia Tech, click here.
Editor’s Note: This article is part of a monthly series that focuses on Campaign Georgia Tech.
Competition may have a high cost for at least one species of tropical seaweed.
Researchers examining the chemical warfare taking place on Fijian coral reefs have found that one species of seaweed increases its production of noxious anti-coral compounds when placed into contact with reef-building corals. But as it competes chemically with the corals, the seaweed grows more slowly and becomes more attractive to herbivorous fish, which boost their consumption of the skirmishing seaweed by 80 percent.
This appears to be the first demonstration that seaweeds can boost their chemical defenses in response to competition with corals. However, determining whether such responses are common or rare awaits additional studies with a broader range of seaweeds and corals.
The research, sponsored by the National Science Foundation and the National Institutes of Health, was published January 8, 2014, in the journal Proceedings of the Royal Society B.
“The important takeaway is that competition between corals and seaweeds can cause dramatic changes in seaweed physiology, both in terms of their growth and their defense,” said Douglas Rasher, who was a graduate student at the Georgia Institute of Technology when the research was conducted. “These changes have potentially cascading effects throughout the rest of the reef community.”
Rasher, now a postdoctoral research associate at the Darling Marine Center at the University of Maine, conducted the research in collaboration with Mark Hay, a professor in the Georgia Tech School of Biology. Hay and Rasher have used coral reefs as field laboratories, studying the chemical signaling that occurs during coral-seaweed competition, and evaluating how herbivorous fish affect the interactions – and long-term health of reefs.
“We previously found that chemical warfare is fairly common among seaweeds and corals, and that several seaweed species are particularly harmful to corals,” Rasher said. “This research explored the degree to which seaweed allelopathy – chemical warfare – is dynamic, how it changes in response to competition, and also whether competition changes the efficacy of other seaweed defenses used against herbivores.”
The findings may also challenge the popular notion that plants cannot change rapidly and strategically in response to their environments.
“We tend to think of plants as being fixed in their behavior,” said Hay. “In fact, plants such as these seaweeds assess their environment continuously, altering biochemically what they are doing as they compete with the coral. These algae somehow sense what is happening and respond accordingly. They may appear passive, but they are really the tricky chemical assassins of coral reefs.”
For this study, Rasher and Hay selected two seaweed species, one (Galaxaura filamentosa) known for its toxicity to corals, and the other (Sargassum polycystum), which does not chemically damage corals. They fragmented pieces of a common coral, Porites cylindrica, glued them into cement cones and placed them on a rack on a reef located in the shallow ocean off the Fiji Islands. The fragments were allowed to grow in the racks for two years.
At the start of the experiment, the researchers took half of the coral samples and dipped them into bleach to kill the living organisms, leaving only the calcium carbonate skeletons. The skeletons served as the control group for the experiments that followed.
The researchers collected samples from both species of seaweed, and split each sample in two. One half of each sample was assigned to a treatment group, while the other half went to the control group. The treatment group was placed into contact with living corals, while the control group was placed into contact with coral skeletons.
The seaweeds were then allowed to interact with the corals and coral skeletons for eight days. After that, a portion of each sample was removed and chemical compounds extracted from them and embedded into small gel strips that were then adhered to other living corals to assess the toxicity of the compounds. The researchers repeated the experiment, placing entire seaweeds in contact with corals to determine if the plants displayed the same effect.
“We saw that Galaxaura, the chemically rich seaweed and the species we knew was allelopathic, had up-regulated its chemistry to become more potent – nearly twice as damaging – when it was in contact with the living coral, compared to those individuals that had only been in contact with the coral skeletons,” Rasher said.
None of the extracts from the Sargassum damaged the corals.
Until this point, the seaweeds and corals had been protected from herbivorous fishes. The next step was to place seaweed samples – both those that had competed with the living coral and those that hadn’t – onto nylon ropes in a location accessible to fish. The researchers created 15 pairs of these samples and placed them at different reef locations.
“We saw that for the non-allelopathic seaweed, Sargassum, fishes didn’t differentiate – they consumed both the treatment and control seaweeds at equal rates,” Rasher said. “But given the option to choose between treatment and control Galaxaura, fishes consumed 80 percent more of the seaweed portions that had been in contact with a living coral.”
The researchers don’t know all the factors that may have made the chemically noxious seaweed more palatable to the fish. However, those seaweed portions that had been competing with coral had less effective chemical defenses against fish. When the researchers took extracts from treatment seaweed and control seaweed and applied them to a palatable seaweed species not previously used in the experiment, fish preferred the seaweed coated with extracts from the portions that had been competing with corals, indicating that competition had compromised the seaweed’s chemical defenses against herbivores.
For the future, the researchers want to study chemical defenses in other seaweeds to determine if what they’ve seen is common among tropical seaweeds that engage in chemical warfare. For now, they don’t know if the chemical defenses evolved to compete with coral or perhaps for another reason, such as fighting off harmful microbes.
The fact that corals may cause seaweeds to up-regulate their anti-coral defenses could help explain why coral reefs rarely bounce back once they begin a decline and become dominated by seaweeds. The research also demonstrates the importance of studying broad interactions among numerous species within complex communities like coral reefs.
“These kinds of interactions show a mechanism that, once the reef begins to crash, could help maintain that decline,” Hay said. “There may be insights here that we could use to better manage, and hopefully restore, some of these systems. We are also hoping that what we learn may bleed over into other systems.”
This research was supported by the National Science Foundation (NSF) under award (OCE-0929119), by the National Institutes of Health (NIH) under award (U01-TW007401), and by the Teasley Endowment to Georgia Tech. The conclusions or recommendations contained in this news release are those of the authors and do not necessarily represent the official positions of the NSF or NIH.
CITATION: Douglas B. Rasher and Mark E. Hay, “Competition induces allelopathy but suppresses growth and anti-herbivore defense in a chemically rich seaweed,” (Proceedings of the Royal Society B, January 2014). http://dx.doi.org/10.1098/rspb.2013.2615
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Taking a DNA molecule into the vicinity of a homologous target gene by a DNA aptamer provides a many-fold enhancement of gene correction frequency at that genetic locus. Aptamer-guided gene targeting, or AGT, is a novel approach for genetic engineering developed by Patrick Ruff in Francesca Storici’s group.
Gene targeting is a genetic technique to modify an endogenous DNA sequence at will, by changing a mutant DNA sequence into a wild-type copy or vice versa in its genomic location via homologous recombination. Gene targeting is therefore a fundamental process not only for functional analysis of genes, proteins, and complex biological systems, but potentially also in molecular therapy for the prevention and cure of human genetic diseases originating from specific DNA alterations. However, editing of genetic information is a challenging task. The goal of gene correction goes far beyond the process of making a desired change in a chosen target gene in the most efficient way. It is essential that the product of the modified gene should then be functional, the DNA correction stable, and the engineering process accurate and restrained to the target in order to minimize unwanted DNA, cellular, and/or tissue damage.
In the most recent years a lot of progress has been made in activating cellular DNA repair and recombination machinery at the target sites for gene correction, mainly via the specific induction of DNA double-strand breaks (DSBs) at these sites. However, there has been much less focus on the other essential component for gene targeting: the donor DNA necessary to make the desired modification. To address the problem of donor DNA availability, Patrick Ruff, fresh PhD recipient in the lab of Francesca Storici from the School of Biology at Georgia Tech, developed a novel gene targeting approach, aptamer-guided gene targeting (AGT), in which he bound the homing endonuclease I-SceI by a DNA aptamer fused to the donor DNA of choice, to target the donor DNA to a desired genetic locus located next to an I-SceI cut site. DNA aptamers, which mimic antibodies, are sequences of DNA that are able to bind to a specific target with high affinity because of their unique secondary structure. Using a variant of capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX) called “Non-SELEX”, Patrick obtained a DNA aptamer for the I-SceI endonuclease, and with the assistance of Storici lab graduate students Kyung Duk Koh and Havva Keskin, and the research scientist Rekha Pai, found that the AGT approach increases the efficiency of gene targeting by guiding an exogenous donor DNA into the vicinity of the site targeted for genetic modification. Dr. Storici said: "by utilizing DNA oligodeoxyribonucleotides that contained the I-SceI aptamer sequence as well as homology to repair the I-SceI DSB and correct a target gene, we were able to increase gene targeting frequencies up to 32-fold over a non-binding control in yeast and up to 16-fold over a non-binding control in human cells".
This study shows that DNA aptamers can be exploited to increase donor DNA availability, and thus promote the transfer of genetic information from a donor DNA molecule to a desired genetic locus. The AGT strategy offers a novel way to increase gene targeting efficiency, represents the first investigation to use aptamers in the context of gene correction, and provides a new direction to the field of genetic engineering.
The study is just published as an article in the journal Nucleic Acids Res (Wednesday February 5, 2014):
Ruff, P., Koh K.D., Keskin H., Pai R.B. and Storici, F. Aptamer-guided gene targeting in yeast and human cells, Nucleic Acids Res, Feb 5 2014 doi:10.1093/nar/gku101 http://nar.oxfordjournals.org/cgi/reprint/gku101?
This project was supported by the Georgia Tech Fund for Innovation in Research and Education (GTFIRE-021763), the NIH grant (R21EB9228), and the Georgia Cancer Coalition grant (award R9028).