Eric Greene found a way to ask what happens when one protein rams into another. "These types of collisions happen all the time in our cells," he says. "But we have a very limited understanding of how they are resolved."

Corralling Lipids

As a postdoc at the National Institutes of Health in the late 1990s, Greene learned how to watch the dynamics of DNA-bound proteins under the microscope. The ability to see the physical wrangling at such a small scale fascinated him. But limitations of the technique—which involved essentially dumping proteins into a vat of DNA and watching one at a time—became frustrating.

"I'd spend hours doing tedious manipulations," says Greene, "and at the end of the day have only one or two data points. This really limited what we could do. It often took weeks or months just to figure out if an experiment was even working." Moreover, in many instances the proteins would stick to the microscope slide, and this nonspecific sticking was a huge problem in the field. Greene struggled to find a surface that worked for all his experiments.

By the time he started his own lab at Columbia, Greene had a plan. He realized that one surface was natural to all proteins: the lipid bilayer, which surrounds every living cell. Greene figured that a bilayer would provide an inert environment to prevent proteins from sticking to a glass slide and also a way to tether DNA in place.

The crux of his method lies in the ability to corral lipids into separate areas of a glass slide. He scratches miniscule lines into the glass, which keep lipids in separate areas—they can't move across the blemishes. He then encloses special lipids, tagged with a molecule called biotin, in one linear area. Unmarked lipids cover the rest of the glass slide. Specially engineered strings of DNA attach only to the biotin-marked lipids, so the ends of the DNA are arranged in a line. Finally, Greene turns on a flow of liquid across the surface, sweeping all the strands in the same direction, forming a curtain of aligned DNA.

This movie shows a DNA curtain (green) bound by fluorescently tagged nucleosomes (magenta). When a constant flow of liquid runs through the setup, the DNA extends in parallel lines, letting researchers see the exact placement of the nucleosomes. When the flow lets up, as seen at 7.4 seconds on the timer, the DNA snaps back up into a more relaxed conformation—until the flow resumes.
Video: Greene Lab

The first time Greene tried this technique, he used a razor blade to scratch the glass—a rudimentary trial, but a successful one. Now, his lab uses electron-beam lithography, a technology developed for manufacturing electronic circuits, to make precisely controlled barriers on the glass that can be used to define the organization of thousands of DNA molecules.

Crash Test Dummies

In principle, the technique behind DNA curtains can be used to study any kind of linear molecule. A scientist could tether RNA, or long filaments that act as cellular highways, to the lipids. But Greene's passion is understanding how proteins interact with DNA. "These relatively simple interactions can drive what an organism looks like, what an organism does, or how it evolves," says Greene. "It's always fascinated me."

Some of the most important protein complexes on DNA are the nucleosomes, the packaging units that consist of DNA wrapped tightly around a protein core. Once Greene established that his curtains worked, he wanted to know whether they would still work for DNA wrapped around nucleosomes.

Greene and his team added nucleosomes to DNA before lining up the strands. When the strands were aligned, the scientists saw that nucleosomes are static once they've attached to DNA. Moreover, by recording the positions of thousands of nucleosomes at one time, Greene's lab had a powerful case for understanding where on DNA nucleosomes attach. The results appeared in the October 2009 issue of Nature Structural & Molecular Biology. The experiment was the first proof of concept that the technique worked on DNA associated with nucleosomes, says Greene.

Photo: Elizabeth Weinberg

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