Whether it is between people or molecules, a handshake can reveal a lot. It shows firmness, indecision, or—as one song lyric put it—a resolve to “hang on loosely” so as not to “lose control.” It can even reveal membership in a sort of conspiracy. In the case of DNA and its binding partners, conspiracies can sway gene expression one way or another.

To divine the outcome of meetings between DNA and DNA-binding proteins, suggests a team of scientists based at Stanford University, researchers should take advantage of a new imaging technique. It combines superresolution microscopy and the simultaneous measurement of single-molecule orientation. It can, in a sense, make out DNA’s secret handshakes.

When people share a secret handshake, they orient their fingers or thumbs in particular positions. Those “in the know” will recognize these subtleties, whereas others will see only a normal handshake. By developing the new imaging technique, the Stanford scientists intend to let researchers be in the know when it comes to DNA.

The new technique was described June 17 in the journal Optica, in an article entitled, “Enhanced DNA Imaging Using Super-Resolution Microscopy and Simultaneous Single-Molecule Orientation Measurements.” The article describes a technique that builds on single-molecule microscopy by adding information about the orientation and movement of fluorescent dyes that attach to DNA.

The Stanford team was led by W.E. Moerner, Ph.D., the founder of single-molecule spectroscopy, a breakthrough method from 1989 that allowed scientists to visualize single molecules with optical microscopy for the first time. Of the 2014 Nobel Laureates for optical microscopy beyond the diffraction limit (William E. Moerner, Stefan W. Hell, and Eric Betzig), Moerner and Betzig used single molecules to image a dense array of molecules at different times.

“You can think of these new measurements as providing little double-headed arrows that show the orientation of the molecules attached along the DNA strand,” said Dr. Moerner. “This orientation information reports on the local structure of the DNA bases because they constrain the molecule. If we didn't have this orientation information the image would just be a spot.”

Single-molecule microscopy, together with fluorescent dyes that attach to DNA, can be used to visualize DNA strands better. Until now, it was difficult to understand how those dyes were oriented and impossible to know if the fluorescent dye was attached to DNA in a rigid or somewhat loose way.

Adam S. Backer, Ph.D., first author of the paper, developed a fairly simple way to obtain orientation and rotational dynamics from thousands of single molecules in parallel. “Our new imaging technique examines how each individual dye molecule labeling the DNA is aligned relative to the much larger structure of DNA,” he explained. “We are also measuring how wobbly each of these molecules is, which can tell us whether this molecule is stuck in one particular alignment or whether it flops around over the course of our measurement sequence.”

The new technique offers more detailed information than today's so-called “ensemble” methods, which average the orientations for a group of molecules, and it is much faster than confocal microscopy techniques, which analyze one molecule at a time. The new method can even be used for molecules that are relatively dim.

Because the technique provides nanoscale information about the DNA itself, it could be useful for monitoring DNA conformational changes or damage to a particular region of the DNA, which would show up as changes in the orientation of dye molecules. It could also be used to monitor interactions between DNA and proteins, which drive many cellular processes.

“We report a simple, high-throughput technique for measuring the azimuthal orientations and rotational dynamics of single fluorescent molecules, which is compatible with localization microscopy,” wrote the authors of the Optica article. “To demonstrate our approach, we use intercalating and groove-binding dyes to obtain super-resolved images of stretched DNA strands through binding-induced turn-on of fluorescence. By combining our image data with thousands of dye molecule orientation measurements, we develop a means of probing the structure of individual DNA strands, while also characterizing dye-DNA interactions.”

To acquire single-molecule orientation information, the researchers used a well-studied technique that adds an optical element called an electro-optic modulator to the single-molecule microscope. For each camera frame, this device changed the polarization of the laser light used to illuminate all the fluorescent dyes.

Because fluorescent dye molecules with orientations most closely aligned with the laser light's polarization will appear brightest, measuring the brightness of each molecule in each camera frame allowed the researchers to quantify orientation and rotational dynamics on a molecule-by-molecule basis. Molecules that switched between bright and dark in sequential frames were rigidly constrained at a particular orientation whereas those that appeared bright for sequential frames were not rigidly holding their orientation.

“If someone has a single-molecule microscope, they can perform our technique pretty easily by adding the electro-optic modulator,” said Backer. “We've used fairly standard tools in a slightly different way and analyzed the data in a new way to gain additional biological and physical insight.”

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