Tagged: DNA Toggle Comment Threads | Keyboard Shortcuts

  • richardmitnick 5:55 pm on October 16, 2014 Permalink | Reply
    Tags: , , , , DNA   

    From BNL: “Scientists Map Key Moment in Assembly of DNA-Splitting Molecular Machine” 

    Brookhaven Lab

    October 15, 2014
    Justin Eure, (631) 344-2347 or Peter Genzer, (631) 344-3174

    The proteins that drive DNA replication—the force behind cellular growth and reproduction—are some of the most complex machines on Earth. The multistep replication process involves hundreds of atomic-scale moving parts that rapidly interact and transform. Mapping that dense molecular machinery is one of the most promising and challenging frontiers in medicine and biology.

    Now, scientists have pinpointed crucial steps in the beginning of the replication process, including surprising structural details about the enzyme that “unzips” and splits the DNA double helix so the two halves can serve as templates for DNA duplication.

    The research combined electron microscopy, perfectly distilled proteins, and a method of chemical freezing to isolate specific moments at the start of replication. The study—authored by scientists from the U.S. Department of Energy’s Brookhaven National Laboratory, Stony Brook University, Cold Spring Harbor Laboratory, and Imperial College, London—published on Oct. 15, 2014, in the journal Genes and Development.

    “The genesis of the DNA-unwinding machinery is wonderfully complex and surprising,” said study coauthor Huilin Li, a biologist at Brookhaven Lab and Stony Brook University. “Seeing this helicase enzyme prepare to surround and unwind the DNA at the molecular level helps us understand the most fundamental process of life and how that process might go wrong. Errors in copying DNA are found in certain cancers, and this work could one day help develop new treatment methods that stall or break dangerous runaway machinery.”

    The research picks up where two previous studies by Li and colleagues left off. They first determined the structure of the “Origin Recognition Complex” (ORC), a protein that identifies and attaches to specific DNA sites to initiate the entire replication process. The second study revealed how the ORC recruits, cracks open, and installs a crucial ring-shaped protein structure (Mcm2-7) that lies at the core of the helicase enzyme.

    But DNA replication is a bi-directional process with two helicases moving in opposite directions. The key question, then, was how does a second helicase core get recruited and loaded onto the DNA in the opposite orientation of the first?

    dr
    Three-dimensional model (based on electron microscopy data) of the double-ring structure loaded onto a DNA helix.

    “To our surprise, we found an intermediate structure with one ORC binding two rings,” said Brookhaven Lab biologist and lead author Jingchuan Sun. “This discovery suggests that a single ORC, rather than the commonly believed two-ORC system, loads both helicase rings.”

    One step further along, the researchers also determined the molecular architecture of the final double-ring structure left behind after the ORC leaves the system, offering a number of key biological insights.

    “We now have clues to how that double-ring structure stably lingers until the cell enters the DNA-synthesis phase much later on in replication,” said study coauthor Christian Speck of Imperial College, London. “This study revealed key regulatory principles that explain how the helicase activity is initially suppressed and then becomes reactivated to begin its work splitting the DNA.”

    three
    Precision methods, close collaboration
    Collaborating scientists and study coauthors Zuanning Yuan of Stony Brook University (standing), Huilin Li of Stony Brook and Brookhaven Lab (seated, back), and Jingchuan Sun of Brookhaven Lab (seated, front) examining protein structures.

    Examining these fleeting molecular structures required mastery of biology, chemistry, and electron microscopy techniques.

    “This three-way collaboration took advantage of each lab’s long standing collaboration and expertise,” said study coauthor Bruce Stillman of Cold Spring Harbor. “Imperial College and Cold Spring Harbor handled the challenging material preparation and functional characterization, while Brookhaven and Stony Brook led the sophisticated molecular imaging and three-dimensional image reconstruction.”

    The researchers used proteins from baker’s yeast—a model organism for the more complex systems found in animals. The scientists isolated the protein mechanisms involved in replication and removed structures that might otherwise complicate the images.

    Once the isolated proteins were mixed with DNA, the scientists injected chemicals to “freeze” the binding and recruitment process at intervals of 2, 7, and 30 minutes.

    They then used an electron microscope at Brookhaven to pin down the exact structures at each targeted moment in a kind of molecular time-lapse. Rather than the light used in a traditional microscope, this technique uses focused beams of electrons to illuminate a sample and form images with atomic resolution. The instrument produces a large number of two-dimensional electron beam images, which a computer then reconstructs into three-dimensional structure.

    “This technique is ideal because we’re imaging relatively massive proteins here,” Li said. “A typical protein contains three hundred amino acids, but these DNA replication mechanisms consist of tens of thousands of amino acids. The entire structure is about 20-nanometers across, compared to 4 nanometers for an average protein.”

    Unraveling the DNA processes at the most fundamental level, the focus of this team’s work, could have far-reaching implications.

    “The structural knowledge may help others engineer small molecules that inhibit DNA replication at specific moments, leading to new disease prevention or treatment techniques,” Li said.

    Additional collaborators on this research include Alejandra Fernandez, Alberto Riera, and Silvia Tognetti of the MRC Clinical Science Centre of Imperial College, London; and Zuanning Yuan of Stony Brook University.

    The research was funded by the National Institutes of Health (GM45436, GM74985) and the United Kingdom Medical Research Council.

    See the full article here.

    BNL Campus

    One of ten national laboratories overseen and primarily funded by the Office of Science of the U.S. Department of Energy (DOE), Brookhaven National Laboratory conducts research in the physical, biomedical, and environmental sciences, as well as in energy technologies and national security. Brookhaven Lab also builds and operates major scientific facilities available to university, industry and government researchers. The Laboratory’s almost 3,000 scientists, engineers, and support staff are joined each year by more than 5,000 visiting researchers from around the world.Brookhaven is operated and managed for DOE’s Office of Science by Brookhaven Science Associates, a limited-liability company founded by Stony Brook University, the largest academic user of Laboratory facilities, and Battelle, a nonprofit, applied science and technology organization.
    i1

    ScienceSprings relies on technology from

    MAINGEAR computers

    Lenovo
    Lenovo

    Dell
    Dell

     
  • richardmitnick 8:43 pm on October 3, 2014 Permalink | Reply
    Tags: , , DNA, ,   

    From LBL: “News Center RCas9: A Programmable RNA Editing Tool” 

    Berkeley Logo

    Berkeley Lab

    October 3, 2014
    Lynn Yarris (510) 486-5375

    A powerful scientific tool for editing the DNA instructions in a genome can now also be applied to RNA, the molecule that translates DNA’s genetic instructions into the production of proteins. A team of researchers with Berkeley Lab and the University of California (UC) Berkeley has demonstrated a means by which the CRISPR/Cas9 protein complex can be programmed to recognize and cleave RNA at sequence-specific target sites. This finding has the potential to transform the study of RNA function by paving the way for direct RNA transcript detection, analysis and manipulation.

    sch
    Schematic shows how RNA-guided Cas9 working with PAMmer can target ssRNA for programmable, sequence-specific cleavage.

    Led by Jennifer Doudna, biochemist and leading authority on the CRISPR/Cas9 complex, the Berkeley team showed how the Cas9 enzyme can work with short DNA sequences known as “PAM,” for protospacer adjacent motif, to identify and bind with specific site of single-stranded RNA (ssRNA). The team is designating this RNA-targeting CRISPR/Cas9 complex as RCas9.

    “Using specially designed PAM-presenting oligonucleotides, or PAMmers, RCas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, or it can be used to isolate specific endogenous messenger RNA from cells,” says Doudna, who holds joint appointments with Berkeley Lab’s Physical Biosciences Division and UC Berkeley’s Department of Molecular and Cell Biology and Department of Chemistry, and is also an investigator with the Howard Hughes Medical Institute (HHMI). “Our results reveal a fundamental connection between PAM binding and substrate selection by RCas9, and highlight the utility of RCas9 for programmable RNA transcript recognition without the need for genetically introduced tags.”

    jd
    Biochemist Jennifer Doudna is leading authority on the CRISPR/Cas9 complex (Photo by Roy Kaltschmidt)

    From safer, more effective medicines and clean, green, renewable fuels, to the clean-up and restoration of our air, water and land, the potential is there for genetically engineered bacteria and other microbes to produce valuable goods and perform critical services. To exploit the vast potential of microbes, scientists must be able to precisely edit their genetic information.

    In recent years, the CRISPR/Cas complex has emerged as one of the most effective tools for doing this. CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is a central part of the bacterial immune system and handles sequence recognition. Cas9 – Cas stands for CRISPR-assisted – is an RNA-guided enzyme that handles the sniping of DNA strands at the specified sequence site.

    Together, CRISPR and Cas9 can be used to precisely edit the DNA instructions in a targeted genome for making desired types of proteins. The DNA is cut at a specific location so that old DNA instructions can be removed and/or new instructions inserted.

    Until now, it was thought that Cas9 could not be used on the RNA molecules that transcribe those DNA instructions into the desired proteins.

    “Just as Cas9 can be used to cut or bind DNA in a sequence-specific manner, RCas9 can cut or bind RNA in a sequence-specific manner,” says Mitchell O’Connell, a member of Doudna’s research group and the lead author of a paper in Nature that describes this research titled Programmable RNA recognition and cleavage by CRISPR/Cas9. Doudna is the corresponding author. Other co-authors are Benjamin Oakes, Samuel Sternberg, Alexandra East Seletsky and Matias Kaplan.

    two
    Benjamin Oakes and Mitch O’Connell are part of the collaboration led by Jennifer Doudna that showed how the CRISPR/Cas9 complex can serve as a programmable RNA editor. (Photo by Roy Kaltschmidt)

    In an earlier study, Doudna and her group showed that the genome editing ability of Cas9 is made possible by presence of PAM, which marks where cutting is to commence and activates the enzyme’s strand-cleaving activity. In this latest study, Doudna, Mitchell and their collaborators show that PAMmers, in a similar manner, can also stimulate site-specific endonucleolytic cleavage of ssRNA targets. They used Cas9 enzymes from the bacterium Streptococcus pyogenes to perform a variety of in vitro cleavage experiments using a panel of RNA and DNA targets.

    “While RNA interference has proven useful for manipulating gene regulation in certain organisms, there has been a strong motivation to develop an orthogonal nucleic-acid-based RNA-recognition system such as RCas9,” Doudna says. “The molecular basis for RNA recognition by RCas9 is now clear and requires only the design and synthesis of a matching guide RNA and complementary PAMmer.”

    The researchers envision a wide range of potential applications for RCas9. For example, an RCas9 tethered to a protein translation initiation factor and targeted to a specific mRNA could essentially act as a designer translation factor to “up-” or “down-” regulate protein synthesis from that mRNA.

    “Tethering RCas9 to beads could be used to isolate RNA or native RNA–protein complexes of interest from cells for downstream analysis or assays,” Mitchell says. “RCsa9 fused to select protein domains could promote or exclude specific introns or exons, and RCas9 tethered to a fluorescent proteins could be used to observe RNA localization and transport in living cells.”

    This research was primarily supported by the NIH-funded Center for RNA Systems Biology.

    See the full article here.

    A U.S. Department of Energy National Laboratory Operated by the University of California

    University of California Seal

    DOE Seal

    ScienceSprings relies on technology from

    MAINGEAR computers

    Lenovo
    Lenovo

    Dell
    Dell

     
  • richardmitnick 8:50 am on September 1, 2014 Permalink | Reply
    Tags: , , , DNA,   

    From Astrobio: “DNA May Have Had Humble Beginnings As Nutrient Carrier” 

    Astrobiology Magazine

    Astrobiology Magazine

    Sep 1, 2014
    Adam Hadhazy

    New research intriguingly suggests that DNA, the genetic information carrier for humans and other complex life, might have had a rather humbler origin. In some microbes, a study shows, DNA pulls double duty as a storage site for phosphate. This all-important biomolecule contains phosphorus, a sometimes hard-to-get nutrient.

    dna

    Maintaining an in-house source of phosphate is a newfound tactic for enabling microorganisms to eke out a living in harsh environments, according to a new study published in the open-access, peer reviewed scientific journal PLOS ONE. The finding bodes well for life finding a way, as it were, in extreme conditions on worlds less hospitable than Earth.

    The results also support a second insight: DNA might have come onto the biological scene merely as a means of keeping phosphate handy. Only later on in evolutionary history did the mighty molecule perhaps take on the more advanced role of genetic carrier.

    “DNA might have initially evolved for the purpose of storing phosphate, and the various genetic benefits evolved later,” said Joerg Soppa, senior author of the paper and a molecular biologist at Goethe University in Frankfurt, Germany.

    Unraveling life’s origins

    Scientists continue to investigate the development of self-replicating, intricate sets of chemistry — in other words, life — from the chemical compounds thought available on early Earth. Out of this mixture of prebiotic chemicals, two nucleic acids — RNA and DNA — emerged as champions.

    early
    Early Earth, in an artist’s impression, where somehow complex, self-replicating chemistry (in other words, life) emerged. Credit: Peter Sawyer / Smithsonian Institution

    Today, these two types of biomolecules serve as the genetic information carriers for all Earthly biota. RNA on its own suffices for the business of life for simpler creatures, such as some viruses. Complex life, like humans, however, relies on DNA as its genetic carrier.

    Astrobiologists want to understand the origin of DNA and its genetic cousin, RNA, because figuring out how life got started here on Earth is key for gauging if it might ever develop on alien planets.

    Many researchers think RNA must have preceded DNA as the genetic molecule of choice. RNA is more versatile, acting as both genetic code and a catalyst for chemical reactions. Explicating the rise of DNA as a genetic material directly from RNA, however, is tricky. Compared to RNA, DNA needs significantly more supporting players for it to work well in a biological setting.

    “The switch from RNA to DNA is not easy because many additional enzymes are required for DNA genomes,” said Soppa.

    This unclear transition from RNA to DNA opens the door for a precursor to DNA possibly having a more mundane job. The new study offers an attractive explanation: that DNA was a fancy way to store nutrients in cells.

    Phosphate depot?

    DNA is chock-full of phosphate. Cells depend on phosphate to form not only DNA and RNA, but also related genetic machinery, such as the ribosome. Phosphate, furthermore, is a must for building the molecule ATP, life’s energy carrier, as well as fatty membrane molecules, certain phospho-proteins and phospho-sugars, and more.

    ds
    The shores of the Dead Sea, which borders Jordan, Palestine and Israel. As the lowest and saltiest lake in the world, it is home to some extreme creatures. Image Credit: Aaron L. Gronstal

    “Phosphate is important for an immense set of biomolecules,” said Soppa.

    Unfortunately for some microbes, ample phosphate is not always available. For example, in salty, nutrient-poor habitats, such as the Dead Sea in the Middle East, an organism called Haloferax volcanii must regularly “eat” ambient DNA to obtain phosphate (plus some other nutritional goodies, such as nitrogen).

    Notably, H. volcanii can still survive and reproduce when phosphorus, the element needed to make phosphate, is lacking. Somehow, then, the microbe must turn to an inner source of phosphate, for otherwise it should cease to grow.

    In their study, Soppa and colleagues from Germany, the United States and Israel sought out this source. The nature of H. volcanii provided some clues. The organism is classified as archaea, one of the three domains of life, in addition to bacteria and eukarya, the latter encompassing all multicellular organisms, from fungi to fruit flies. Many archaea and bacteria — collectively, “prokaryotes”— have just one, circular chromosome. Eukaryotes, like us, on the other hand, can have any number of the chunky pieces of DNA, RNA and proteins. (Humans have 23 pairs of different chromosomes, for the record.) H. volcanii is unusual. It has 20 copies of the same chromosome when it’s growing happily under favorable conditions, and 10 when nutrients are exhausted and it reaches a stationary phase.

    Strength in numbers

    Lots of chromosome copies are good to have in a pinch. So-called polyploidal organisms like H. volcanii use their copious chromosomes to tough it out through bad situations, such as high radiation exposure or total dry-outs, called desiccation. Either scenario causes the strands in chromosomal DNA to break. For single-chromosome species, only a few breaks lead to death because it is impossible to repair a chromosome scattered into fragments.

    But if there are multiple copies of the cracked chromosomes, fragments can fortuitously line up. Rather like how a jigsaw puzzle is easier to put together if there are numerous duplicates of each necessary piece, the chromosome shards can sync up and restore a functional chromosome.

    hv
    H. Volcanii grown in culture. Credit: Yejineun/Wikipedia

    “In polyploid species, the fragments generated from different copies of the chromosome overlap, and it is possible to regenerate an intact chromosome from overlapping fragments,” said Soppa.

    Desperate times, desperate measures

    To investigate if H. volcanii‘s extra chromosomes might help the archaeon survive low phosphate conditions, Soppa and colleagues starved the organism in the lab of the critical substance. The microbe continued to reproduce by splitting one cell apart into two. Interestingly, chromosome counts diminished in the “parent” and the “daughter” cells.

    “From quantifying the number of chromosomes prior to and after growth in the absence of phosphate, we have found that about 30 percent of the chromosomes are ‘missing’ afterwards,” said Soppa.

    The numbers for another potential in-house source of phosphate, H. volcanii‘s ribosomes, however, remained steady. The most likely explanation, then, of the microorganism’s hardiness when facing a phosphate nutrient shortage: H. volcanii simply cannibalizes some of its own chromosomes.

    As further verification, Soppa and colleagues tested the survival skills of H. volcanii cells that contained varying numbers of chromosome copies. Those archaea with just two copies of their chromosome turned out to be more than five times as sensitive to desiccation as those H. volcanii with a hefty complement of 20 chromosomes.

    Life, undaunted

    This newly described benefit of polyploidy in H. volcanii is a fresh demonstration of how life can make do in severe environments. So-called extremophiles have been discovered in recent decades thriving in strongly acidic hot springs, within liquid asphalt, and in other eyebrow-raising niches. Salt-tolerant bacteria and archaea, like H. volcanii, have been found to survive in deserts, simulated Mars conditions, and even the rigors of a space flight. We should not be surprised, perhaps, if life has managed to take hold on formidable worlds.

    rw
    Extremophile microbes have been found that can survive in the polluted Rio Tinto River in Spain. Mining in the river’s vicinity has led to its waters having a high heavy metal content and very low pH, though the bacteria themselves, through their metabolism, also likely contribute to the intense acidity. Image credit: Leslie Mullen

    “The understanding of how harsh the conditions can be that can be survived by some archaea and bacteria helps us to be more optimistic that life could have evolved at very rough and unsuitable places on early Earth or on other planets,” said Soppa.

    The new role ascribed to DNA, as phosphate storage, might help to explain how a completely RNA-dominated primordial era began sharing genetic duties with DNA. Life did not leap from RNA to DNA. Rather, DNA, slowly but surely, learned new tricks.

    “The hypothesis that DNA might have evolved as a storage polymer and became genetic material later, makes the step from RNA to DNA as genetic material easier, because it then would be a two-step and not a one-step process,” said Soppa. “DNA would have been around, and during long time spans additional roles could have been evolved.”

    See the full article here.
    Astrobiology Magazine is a NASA-sponsored online popular science magazine. Our stories profile the latest and most exciting news across the wide and interdisciplinary field of astrobiology — the study of life in the universe. In addition to original content, Astrobiology Magazine also runs content from non-NASA sources in order to provide our readers with a broad knowledge of developments in astrobiology, and from institutions both nationally and internationally. Publication of press-releases or other out-sourced content does not signify endorsement or affiliation of any kind.
    Established in the year 2000, Astrobiology Magazine now has a vast archive of stories covering a broad array of topics.

    NASA

    ScienceSprings relies on technology from

    MAINGEAR computers

    Lenovo
    Lenovo

    Dell
    Dell

     
  • richardmitnick 6:07 pm on June 23, 2014 Permalink | Reply
    Tags: , , , DNA, ,   

    From SLAC Lab: “Scientists Use X-rays to Look at How DNA Protects Itself from UV Light” 


    SLAC Lab

    June 23, 2014
    Andrew Gordon, agordon@slac.stanford.edu, (650) 926-2282

    The molecular building blocks that make up DNA absorb ultraviolet light so strongly that sunlight should deactivate them – yet it does not. Now scientists have made detailed observations of a “relaxation response” that protects these molecules, and the genetic information they encode, from UV damage.

    The experiment at the Department of Energy’s SLAC National Accelerator Laboratory focused on thymine, one of four DNA building blocks. Researchers hit thymine with a short pulse of ultraviolet light and used a powerful X-ray laser to watch the molecule’s response: A single chemical bond stretched and snapped back into place within 200 quadrillionths of a second, setting off a wave of vibrations that harmlessly dissipated the destructive UV energy.

    The international research team reported the results June 23 in Nature Communications.

    While protecting the genetic information encoded in DNA is vitally important, the significance of this result goes far beyond DNA chemistry, said Philip Bucksbaum, director of the Stanford PULSE Institute and a co-author of the report.

    “The new tool the team developed for this study provides a new window on the motion of electrons that control all of chemistry,” he said. “We think this will enhance the value and impact of X-ray free-electron lasers for important problems in biology, chemistry and physics.”

    Light Becomes Heat

    Researchers had noticed years ago that thymine seemed resistant to damage from UV rays in sunlight, which cause sunburn and skin cancer. Theorists proposed that thymine got rid of the UV energy by quickly shifting shape. But they differed on the details, and previous experiments could not resolve what was happening.

    The SLAC experiment took place at the Linac Coherent Light Source (LCLS), a DOE Office of Science user facility, whose bright, ultrashort X-ray laser pulses can see changes taking place at the level of individual atoms in quadrillionths of a second.

    Scientists turned thymine into a gas and hit it with two pulses of light in rapid succession: first UV, to trigger the protective relaxation response, and then X-rays, to detect and measure the response.

    “As soon as the thymine swallows the light, the energy is funneled as quickly as possible into heat, rather than into making or breaking chemical bonds,” said Markus Guehr, a DOE Early Career Program recipient and senior staff scientist at PULSE who led the study. “It’s like a system of balls connected by springs; when you elongate that one bond between two atoms and let it loose, the whole molecule starts to tremble.”

    Ejected Electrons Signal Changes

    The X-rays measured the relaxation response indirectly by stripping away some of the innermost electrons from atoms in the thymine molecule. This sets off a process known as Auger decay that ultimately ejects other electrons. The ejected electrons fly into a detector, carrying information about the nature and state of their home atoms.

    By comparing the speeds of the ejected electrons before and after thymine was hit with UV, the researchers were able to pinpoint rapid changes in a single carbon-oxygen bond: It stretched when hit with UV light and shortened 200 quadrillionths of a second later, setting off vibrations that continued for billionths of a second.

    “This is the first time we’ve been able to distinguish between two fundamental responses in the molecule – movements of the atomic nuclei and changes in the distribution of electrons – and time them within a few quadrillionths of a second,” said the paper’s first author, Brian McFarland, a postdoctoral researcher who has since moved from SLAC to Los Alamos National Laboratory.

    Guehr said the team plans more experiments to further explore the protective relaxation response and extend the new method, called time-resolved Auger spectroscopy, into other scientific realms.

    In addition to the Stanford PULSE Institute, which is a joint institute of SLAC and Stanford University, the study included researchers from LCLS, Stanford, the University of Perugia in Italy, Lawrence Berkeley National Laboratory, the University of Connecticut, Western Michigan University, the University of Gothenburg in Sweden, and UNIST in South Korea. Parts of the research were carried out at Berkeley Lab’s Advanced Light Source, a DOE Office of Science user facility. The work was funded by the DOE Office of Science, the Swedish Research Council, the Göran Gustafsson Foundation and the Knut and Alice Wallenberg Foundation.

    See the full article here.

    SLAC Campus
    SLAC is a multi-program laboratory exploring frontier questions in photon science, astrophysics, particle physics and accelerator research. Located in Menlo Park, California, SLAC is operated by Stanford University for the DOE’s Office of Science.
    i1


    ScienceSprings is powered by MAINGEAR computers

     
  • richardmitnick 9:02 am on April 28, 2014 Permalink | Reply
    Tags: , , DNA   

    From Brookhaven Lab: “Label-free, Sequence-specific, Inexpensive Fluorescent DNA Sensors” 

    Brookhaven Lab

    April 28, 2014
    Karen McNulty Walsh

    Intensity of glow indicates level of genetic match; potentially useful for identifying microbes, harmful agents, and more

    Using principles of energy transfer more commonly applied to designing solar cells, scientists at the U.S. Department of Energy’s Brookhaven National Laboratory have developed a new highly sensitive way to detect specific sequences of DNA, the genetic material unique to every living thing. As described in a paper published in the journal Chemistry of Materials, the method is considerably less costly than other DNA assays and has widespread potential for applications in forensics, medical diagnostics, and the detection of bioterror agents.

    map
    Light-up DNA sensors: When DNA sequences are complementary, one fluorescent dye molecule (red rectangle) binds between each matching base pair. The conjugated polymer (blue) wraps around the DNA, absorbs light, and transfers the energy (via fluorescence resonant energy transfer, or FRET) to the dye to amplify the photo luminescent (PL) signal (red arrow). When DNA strands don’t match perfectly, fewer dye molecules bind and the magnitude of the PL signal is reduced.

    “This system is sensitive enough to detect individual mismatches between the bases that make up the ‘rungs’ of the twisted-ladder DNA double helix molecule, making it highly specific with no false positives.”
    — Mircea Cotlet

    “The sensors we’ve developed use a light-absorbing polymer to amplify the fluorescent signal of a dye that emits light only when it binds between two matched pieces of DNA,” said Mircea Cotlet, a physical chemist at Brookhaven’s Center for Functional Nanomaterials, who led the research and who is also an adjunct professor at Stony Brook University. The system is sensitive enough to detect individual mismatches between the bases that make up the “rungs” of the twisted-ladder DNA double helix molecule, making it highly specific with no false positives, Cotlet said.

    Plus, the method is rapid and requires no expensive equipment, just a conventional laboratory fluorimeter. It has high potential to be made field deployable for rapid analysis of crime-scene evidence and to mount a more knowledgeable, speedy response to bioterror threats.
    DNA, show thyself!

    The idea of using glowing dyes to sniff out DNA sequences is not entirely new. But finding an inexpensive fluorescent dye that inserts itself between every complementary base pair of a DNA molecule—and using a light absorbing/emitting polymer to amplify the fluorescent signal without the need for additional chemical tagging—makes the Brookhaven approach a big advance.

    two
    Zhongwei Liu, a graduate student from Stony Brook University, working with Brookhaven’s Mircea Cotlet on a new type of DNA sensor at the Center for Functional Nanomaterials.

    “The dye we use is hundreds of times cheaper than popular commercial intercalating dyes,” said Zhongwei Liu, a graduate student from Stony Brook University working with Cotlet and first author of the paper. Unbound, the green colored molecule absorbs red light but does not emit light. “But when it inserts itself in the grooves of the DNA, the dye becomes fluorescent. And so far it is the only dye that can intercalate so densely with DNA—meaning exactly one dye molecule binds between each complementary base pair of the DNA double helix”—the T-A and G-C matches that make up the genetic code.

    That means the strength of the fluorescent signal is directly related to how many dye molecules are bound—and how closely an unknown DNA sample matches a probe strand used for testing. As soon as there’s a mismatch, even at just one “rung” on the DNA ladder, a dye molecule won’t bind and the signal will weaken. Two mismatches results in a proportional drop in signal strength, and so on. “That gives us a large range for the detection of sequence mismatch,” Cotlet said.

    To amplify these signals, the scientists add a conjugated polymer. These light-absorbing materials are used for harvesting sunlight in solar cells, “but we can also make them water soluble and compatible with biomolecules like DNA,” Cotlet said.

    Synthesized by Hsing-Lin Wang, a collaborator at DOE’s Los Alamos National Laboratory, the polymers used in this study were functionalized with side chains that carry a positive charge, allowing them to naturally bind with negatively charged DNA via electrostatic interactions. “The polymer wraps and follows the helix of the DNA,” Cotlet said. “This configuration brings the polymer in close proximity with the DNA-bound dye molecules and also enhances the polymer’s ability to absorb and emit light. Both of these factors help with the transfer of energy to the dye-intercalated DNA and increase the sensitivity of the biosensor,” Cotlet said.

    So if scientists want to know whether two pieces of DNA are identical—say a known sequence from an anthrax spore and one from a suspicious-looking white powder—all they have to do is mix the samples, dye, and polymer in a test tube, turn on the light, and let the results shine for themselves. Of course, in this case, they’d be hoping to not see the light!

    This research was supported by the DOE Office of Science.

    See the full article here.

    One of ten national laboratories overseen and primarily funded by the Office of Science of the U.S. Department of Energy (DOE), Brookhaven National Laboratory conducts research in the physical, biomedical, and environmental sciences, as well as in energy technologies and national security. Brookhaven Lab also builds and operates major scientific facilities available to university, industry and government researchers. The Laboratory’s almost 3,000 scientists, engineers, and support staff are joined each year by more than 5,000 visiting researchers from around the world.Brookhaven is operated and managed for DOE’s Office of Science by Brookhaven Science Associates, a limited-liability company founded by Stony Brook University, the largest academic user of Laboratory facilities, and Battelle, a nonprofit, applied science and technology organization.
    i1


    ScienceSprings is powered by MAINGEAR computers

     
  • richardmitnick 4:44 pm on January 29, 2014 Permalink | Reply
    Tags: , DNA, ,   

    From Berkeley Lab: “Puzzling Question in Bacterial Immune System Answered” 


    Berkeley Lab

    January 29, 2014
    Lynn Yarris (510) 486-5375 lcyarris@lbl.gov

    Berkeley Researchers Uncover the Key to Self-Awareness in Genome Editor

    A central question has been answered regarding a protein that plays an essential role in the bacterial immune system and is fast becoming a valuable tool for genetic engineering. A team of researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California (UC) Berkeley have determined how the bacterial enzyme known as Cas9, guided by RNA, is able to identify and degrade foreign DNA during viral infections, as well as induce site-specific genetic changes in animal and plant cells. Through a combination of single-molecule imaging and bulk biochemical experiments, the research team has shown that the genome-editing ability of Cas9 is made possible by the presence of short DNA sequences known as “PAM,” for protospacer adjacent motif.

    dna
    Short DNA sequences known as “PAM” (shown in yellow) enable the bacterial enzyme Cas9 to identify and degrade foreign DNA, as well as induce site-specific genetic changes in animal and plant cells. The presence of PAM is also required to activate the Cas9 enzyme. (Illustration by KC Roeyer.)

    “Our results reveal two major functions of the PAM that explain why it is so critical to the ability of Cas9 to target and cleave DNA sequences matching the guide RNA,” says Jennifer Doudna, the biochemist who led this study. “The presence of the PAM adjacent to target sites in foreign DNA and its absence from those targets in the host genome enables Cas9 to precisely discriminate between non-self DNA that must be degraded and self DNA that may be almost identical. The presence of the PAM is also required to activate the Cas9 enzyme.”

    With genetically engineered microorganisms, such as bacteria and fungi, playing an increasing role in the green chemistry production of valuable chemical products including therapeutic drugs, advanced biofuels and biodegradable plastics from renewables, Cas9 is emerging as an important genome-editing tool for practitioners of synthetic biology.

    “Understanding how Cas9 is able to locate specific 20-base-pair target sequences within genomes that are millions to billions of base pairs long may enable improvements to gene targeting and genome editing efforts in bacteria and other types of cells,” says Doudna who holds joint appointments with Berkeley Lab’s Physical Biosciences Division and UC Berkeley’s Department of Molecular and Cell Biology and Department of Chemistry, and is also an investigator with the Howard Hughes Medical Institute (HHMI).

    two
    Jennifer Doudna and Samuel Sternberg used a combination of single-molecule imaging and bulk biochemical experiments to show how the RNA-guided Cas9 enzyme is able to locate specific 20-base-pair target sequences within genomes that are millions to billions of base pairs long. (Photo by Roy Kaltschmdit)

    Doudna is one of two corresponding authors of a paper describing this research in the journal Nature. The paper is titled DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. The other corresponding author is Eric Greene of Columbia University. Co-authoring this paper were Samuel Sternberg, Sy Redding and Martin Jinek.

    See the full article here.

    A U.S. Department of Energy National Laboratory Operated by the University of California

    University of California Seal

    DOE Seal


    ScienceSprings is powered by MAINGEAR computers

     
c
Compose new post
j
Next post/Next comment
k
Previous post/Previous comment
r
Reply
e
Edit
o
Show/Hide comments
t
Go to top
l
Go to login
h
Show/Hide help
shift + esc
Cancel
Follow

Get every new post delivered to your Inbox.

Join 355 other followers

%d bloggers like this: