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New DNA Imaging Technique Boasts Nanoscale Resolution

NEW YORK (GenomeWeb) – Researchers from Stanford University have developed a new DNA imaging technique based on single-molecule microscopy that allows scientists to view DNA strands at the nanoscale.

"Our new imaging technique examines how each individual dye molecule labeling the DNA is aligned relative to the much larger structure of DNA," Adam Backer, first author and graduate student at Stanford University, said in a statement. "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."

Learning how "wobbly" the dye molecules are helps better inform polarization and therefore orientation data that can reveal more about what is happening to the DNA strand on the nanoscale, the researchers noted.

The researchers believe that this new imaging technique provides nanoscale information about the DNA itself that could be useful for monitoring DNA conformation changes or damage to a particular region of the DNA, which would show up as changes in the orientation of dye molecules. They also believe that this technique could be used to monitor interactions between DNA and proteins, which drive many cellular processes.

In a paper published this week in the journal Optica, the research team described their new technique and demonstrated it by obtaining super-resolution images and orientation measurements for thousands of single fluorescent dye molecules attached to DNA strands.

The researchers modified a well-studied technique, called single-molecule microscopy, and added an optical element called an electro-optic modulator to acquire single-molecule orientation information.The optic modulator allowed the team to change the polarization of the laser light used to illuminate all the fluorescent dyes for each camera frame, which made fluorescent dye molecules that were most closely aligned with the laser light's polarization appear brighter and those far away to appear darker.

When molecules from the images switched between bright and dark in sequential frames, researchers determined that they were rigidly constrained at a particular orientation, while molecules that appeared bright for sequential frames were considered to have looser constraint in orientation.

In the paper, the researchers demonstrated a spatial resolution of around 25 nanometers and single-molecule orientation measurements with an accuracy of around 5 degrees. They also measured the rotational dynamics, or floppiness, of single molecules with an accuracy of about 20 degrees.

However to actually test the method's efficacy in determining orientation information, the research team looked at how effectively it worked using different types of dyes. First, the researchers analyzed an intercalating dye — a fluorescent dye that slides into the areas between DNA bases — called SYTOX Orange. This enabled the acquisition of up to 300,000 single molecule locations and 30,000 single-molecule orientation measurements in just over 13 minutes.

The team's analysis showed that the individual dye molecules were oriented perpendicular to the DNA strand's axis. While the molecules tended to orient in this perpendicular direction, they did move around just within a constrained cone.

Next, the researchers performed a similar analysis using a different type of fluorescent dye called SiR-Hoechst that consists of two parts: one part that attaches to the side of the DNA and a fluorescent part that is connected via a "floppy" tether. They found that the enhanced DNA imaging technique could detect this "floppiness," and noted that the method could be useful in helping scientists understand, on a molecule-by-molecule basis, whether different labels attach to DNA in a mobile or fixed way.

"This work suggests that intercalating dyes may serve as an exquisite sensor for studying DNA-binding proteins at the single-molecule level," the researchers note in the paper. "For example, nucleosome complexes play a vital role in DNA compaction and regulating gene expression by virtue of the manner in which they wrap DNA into 'loops.' The formation of such loops may be readily monitored in real time by using single-molecule orientation data to detect bends in the DNA substrate."