A strand of DNA is a very long, narrow string, just a few nanometers across. Single-molecule orientation measurements provide unparalleled insight into a multitude of biological and polymeric systems. Single-molecule microscopy, together with fluorescent dyes that attach to DNA, can be used to better visualize this tiny string. However, it is difficult to understand how those dyes are oriented and impossible to know if the fluorescent dye was attached to the DNA in a rigid or somewhat loose way. Now, researchers at Stanford University have developed a new enhanced DNA imaging technique that can probe the structure of individual DNA strands at the nanoscale. The team state that since DNA is at the root of many disease processes, the technique could help scientists gain important insights into what goes wrong when DNA becomes damaged or when other cellular processes affect gene expression. The opensource study is published in the journal Optica.
Previous studies show that fluorescence microscope is an effective tool for measuring the orientations of single molecules. By directly measuring the tilt and wobble of individual fluorescent probes, researchers have gained an understanding of the mechanical properties of DNA, the acrobatics of motor proteins, as well as measures of molecular order in single cells, biological filaments, and polymers. However, as yet, researchers have been unable to develop a method to probe the structure of individual DNA strands, whilst characterizing dye-DNA interactions. The current study builds on a technique called single-molecule microscopy by adding information about the orientation and movement of fluorescent dyes attached to the DNA strand.
The current study investigated the enhanced DNA imaging technique by using it to analyze an intercalating dye; a type of fluorescent dye that slides into the areas between DNA bases. Results show that in a typical imaging experiment, 300,000 single molecule locations and 30,000 single-molecule orientation measurements were acquired in just over 13 minutes. Data findings show that the individual dye molecules were oriented perpendicular to the DNA strand’s axis and that while the molecules tended to orient in this perpendicular direction, they also moved around within a constrained cone.
The lab then performed a similar analysis using a different type of fluorescent dye 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. The group observed that the enhanced DNA imaging technique detected this floppiness, meaning that the method could be useful in helping to detect, on a molecule by molecule basis, whether different labels attach to DNA in a mobile or fixed way.
The researchers state that their new technique demonstrated a spatial resolution of around 25 nanometers and single-molecule orientation measurements with an accuracy of around 5 degrees. They also successfully measured the rotational dynamics, or floppiness, of single-molecules with an accuracy of about 20 degrees.
The team surmise that their new technique offers more detailed information than the currently available ‘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. They go on to add that the new method can even be used for molecules that are relatively dim. For the future, the researchers state that 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.
Source: The Optical Society