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These dots are basically fluorescent molecules that are attached to specific features of the DNA and can be used to identify the DNA, its genomic location, and to uncover all the overlaying epigenetic information,” says Dr Ebenstein. This technique could, for example, enable researchers to identify the specific location of epigenetic marks or DNA damage. “We’ve developed a way to fluorescently mark DNA damage. It’s very interesting to know if your DNA is damaged at specific places when you’re exposed to the sun,” continues Dr Ebenstein. “One of the things we’re trying to do is to taint these damaged sites with a specific colour, and when we stretch the DNA and map it, we want to see if we have accumulations of damage at specific locations. So if a cancer patient who is taking a drug, and one of the side-effects is UV sensitivity, then what happens to these cells if they’re exposed to the sun?” This kind of information is not easily accessible with existing sequencing technologies, as they work by looking at the population level. Dr Ebenstein and his colleagues aim to develop a toolbox that allows researchers to see the properties of individual DNA molecules. “We’re sensitive to a lot of information that’s not easily accessible with DNA sequencing. For example, with a single snapshot on the microscope, we can see all the different kinds of information represented with different colours on a DNA molecule,” he explains. Researchers are developing chemoenzymatic approaches to label these different types of information with a different chemical moiety, combining chemistry with enzymology. “Enzymes are natural molecular machines, that have a lot of functions in biology. There are many types of enzymes, and usually they perform a specific task,” outlines Dr Ebenstein. “For example, there is a family

of enzymes called restriction enzymes, which are molecular scissors. They know how to read a certain word on DNA, and cut the DNA at that word. These restriction enzymes evolved to defend bacteria from invading viruses. Once the DNA from the viral genome goes into the bacteria, these molecular scissors identify it, and they cut it up.” The project is using such enzymes in a different way, using their ability to perform sequence specific chemistry on DNA molecules. DNA is a double-stranded molecule, so to cut it you have to cut both strands. These enzymes have been mutated and instead of cutting DNA into two pieces they only cut one strand, leaving a nick. “So these enzymes know how to read a specific word of DNA, and to cut one strand of it. Now, we introduce another enzyme, DNA polymerase. This enzyme repairs DNA – when it sees the nick, it grabs a base from solution and it heals the nick. It introduces it into this gap and fixes the DNA,” explains Dr Ebenstein. Researchers then effectively trick this enzyme, giving it a nucleotide, a base, with a fluorescent molecule attached. “This enzyme is now actually doing the chemistry for us – it’s introducing a fluorescent molecule, a light-emitting molecule, into the exact word where the nicking enzyme has made its nick,” continues Dr Ebenstein. “This system has evolved naturally for millions of years, and is very efficient and very specific. We take this system out of its natural context and use it for a biotechnological application.” Dr. Ebenstein and his team utilize this concept of manipulating the activity of naturally occurring enzymes for labelling various epigenetic marks in the genome, thus expanding the available toolbox for single-molecule genomic barcoding and mapping.

At a glance Full Project Title Experimental Toolbox for Unmasking Genetic / Epigenetic Variation in Genomic DNA and Chromatin (Beads on String Genomics) Project Objectives The ground-breaking goal of this research project is to establish a robust experimental toolbox – ‘beads-onstring’ -for integrated genetic/epigenetic profiling of native chromosomes. A successful accomplishment of this goal will allow the characterization of genomic variation otherwise hidden by ensemble averaging and will open new horizons in genomic research and personalized medicine. Project Funding 1,627,600 euros Contact Details Principle investigator, Dr Yuval Ebenstein School of chemistry Tel Aviv University Israel T: +972-3-6408901 E: W:

Dr Yuval Ebenstein

Yuval Ebenstein studied chemistry and physics at the Hebrew University in Jerusalem, Israel, where he also completed his Ph.D. in physical chemistry with Prof. Uri Banin, studying the photophysical properties of individual semiconductor nanocrystal quantum dots (QDs). He then moved to work as a postdoc with Prof. Shimon Weiss at UCLA where he used QDs to light-up individual DNA binding proteins and map them along bacteriophage genomes. In the summer of 2011 he set-up the NanoBioPhotonix Lab in the department of chemical physics, school of chemistry at Tel Aviv University.