DNA may be one of the most basic molecules of life, but it's not easy to study. Despite being a very long molecule, it's only about 2.2 nanometers wide, so it's hard to see. Attaching fluorescent dyes to it can help, but until now, knowing how those dye molecules were behaving wasn't possible. A team of scientists at Stanford University led by William E. Moerner has built on a technique called "single-molecule microscopy" to see just how DNA-bound dye molecules orient themselves, flop around and glow in the presence of polarized laser light.
Using marker molecules – such as radioactive tracers and fluorescent dyes — to study DNA has been standard practice in biochemistry for decades. It goes a long way toward helping scientists understand how DNA molecules are put together and how they work, but it's a low-resolution technique that shows where something is happening, but not necessarily what.
First developed by Moerner — a chemical physicist at Stanford — in 1989, single-molecule microscopy is now being used by the Stanford team to mark thousands of DNA strands with fluorescent dye molecules and then beam them with polarized laser light to create more informative images than ever before. According to the team, this technique not only marks the position of the marker molecule, but also its orientation and whether the molecule is attached firmly or loosely to the strand, which can reveal information about the underlying DNA.
"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," says 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."
The new technique works by means of an optical element known as an electro-optic modulator, which modulates a laser beam to change its polarity. By shining the laser on a marked DNA strand and shifting its polarity, fluorescent markers pointing in different directions shine at different intensities. By measuring the brightness of each marker molecule, scientists can measure which way it's pointing. In addition, by studying the markers in sequential frames, it's possible to deduce whether they're stuck fast to the DNA strand or wobbling.
Stanford says that the new technique can measure 300,000 single molecule locations and 30,000 single-molecule orientation measurements in a little more than 13 minutes. Using the new technique, a resolution of 25 nanometers and single-molecule orientations of five degrees were obtained, as well as measurements of the floppiness of molecules to within 20 degrees.
According to Stanford, the ability to study DNA at this level will allow researchers to identify changes or damages in DNA strands as well as study how DNA and proteins interact.
The research finds were published in Optica.
Source: The Optical Society