In the past, researchers have been able to view double-stranded DNA under optical microscopes but not single-stranded DNA. Scientists at Rice University in Houston, TX have found a way to localize certain sequences along single-stranded DNA by making use of high-tech optical equipment and advanced mathematics that breaks the diffraction limit of light. This new technique, known as “super-localization microscopy,” might pave the way to diagnosing genetic diseases.
Dr. Christy Landes, a chemist at Rice, and colleagues are working on proof-of-concept experiments that have identified short sequences of DNA (50 nucleotides) at room temperature. This super-resolution technique has been dubbed “motion blur point accumulation for imaging in nanoscale topography” (mbPAINT). The researchers are able to resolve structures as small as 30 nanometers by making a movie of fluorescent DNA markers flowing over a known DNA sequence along a stationary single strand of DNA.
The markers, or probes, contain a fluorescent dye that is activated when they attach to the target DNA. Most of the probes actually flow by unseen, however some bind for a few milliseconds. This is long enough for the image to be caught on camera before the probes move on. Processing images of these short events allows scientists to observe objects that are too small to be seen with natural diffraction of light-based imaging.
Rice postdoctoral associate, Jixin Chen, notes, “The probes are moving so fast that in real time, all we would see with the camera is a line”. However, the camera takes pictures at 30-millisecond intervals and manages to catch a bound probe. On occasion, probes are able to bind to two sequences along a single strand. With regular fluorescent microscopy, this would appear as a single blur.
The researchers hope to use mbPAINT to map smaller fragments of DNA in the future. Landes notes, “Eventually, we’d like to get down to a couple of nucleotides. Some diseases are characterized by one amino acid mutation, which is three nucleotides, and there are many diseases associated with very small genetic mutations that we’d like to be able to identify. We’re thinking this method will be ideally suited for diseases associated with small, localized mutations that are not possible to detect in any other inexpensive way”.
Landes believes that mbPAINT is a cost-effective technique that has the ability to acquire information that electron microscopes can’t. Moreover, this process allows observation of biological processes of nano-sized objects that are in water at room temperature or even body temperature.
The current research is available in the American Chemical Society journal Applied Materials and Interfaces. The authors include Christy Landes, Rice postdoctoral associate Jixin Chen and undergraduate student Alberto Bremauntz.