Scientists improve DNA detection with nanotags

Researchers in the chemistry department, alongside colleagures in the Molecular and Biosensor and Imaging Center (MBIC), have just made the process of viewing and identifying DNA much easier.

MBIC conducted research to improve the process by which scientists identify and distinguish different strands of DNA.

Associate professor of chemistry Bruce Armitage, alongside colleagues from MBIC, recently published an online paper entitled “Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates” in the Journal of the American Chemical Society.

The MBIC, recently named the National Center for Networks and Pathways by the National Institutes of Health, is a center utilized by both Carnegie Mellon and University of Pittsburgh researchers, in addition to a number of other universities. The MBIC has a history of researching and developing fluorescent detection methods by combining fluorescent probes, or areas of cells that produce color, with computerized microscopy.

Armitage’s recent paper outlines a method for improving the process of fluorescent dyeing, which consists of binding fluorescent dye molecules to DNA. Color and concentration variations allow for further levels of strand distinction.

According to Alan Waggoner of MBIC, “The idea is to hook very bright fluorescent tags to things like DNA and proteins so that they can detect it.” Scientists have used fluorescent detection methods in the past to uncover the human genome sequence, monitor AIDS, and diagnose cancer.

A Carnegie Mellon press release published on January 26 stated that this new nanotagging technique is an innovative advancement in detecting rare cancers within biopsy tissue samples. Moreover, monitoring DNA activity of mutant and healthy cells is important for determining the effectiveness of cancer treatments in individual patient cases. The population and activity of cancerous and healthy cells within a tissue can help doctors determine whether recurrence is likely and further treatment is needed.

“For example, two different populations of cells, one healthy and the other cancerous, could be distinguished based on labeling them with different color fluorescent nanotags,” Armitage said in the press release.

Graduate student Andrea Benvin, who is part of Armitage’s research group, stated in the press release, “Our DNA nanotags offer unprecedented densities of fluorescent dyes and, thus, the potential for extremely bright fluorescent labels.... We’ve put it all into a very small package, which will allow us to detect molecules with great sensitivity without interfering with the biological processes we are trying to understand.”

The structure of a fluorescent molecule is based on the design of phycobiliproteins, proteins found in certain types of photosynthetic algae. According to the press release, the proteins contain multiple fluorescent pigments that absorb light energy and transfer it to chlorophyll before photosynthetic processes utilize the absorbed energy. The fluorescent DNA nanotags developed by the MBIC team imitate this light-harvesting process to create very luminescent, fluorescent labels.

According to Armitage, “light-harvesting” tags allow for further differentiation between different DNA strands. These light-harvesting dyes are excited by a certain wavelength of light and transfer energy to other “light-emitting” tags on the nanotag’s surface. The interaction between tags and absorbed light causes the light-emitting and light-harvesting tags to fluoresce at different colors, accounting for the additional capacity to differentiate between strands.

The fluorescent molecules are inserted in between DNA bases, which are stacked on top of one another. Other dyeing methods usually have a single fluorescent marker for every strand of DNA, so this new method far exceeds previous levels of brightness. For example, if a strand of DNA consists of 30 bases, 15 markers can be attached to the molecule as opposed to one. This can create a level of fluorescence 15 times as bright as normal.

According to Armitage’s publication, these assemblies of fluorescent molecules are advantageously simple. In particular, researchers used base pairing to assemble the DNA nanostructure, and the fluorescent molecules were then added to the solution to form non-covalent bonds with the DNA.

In the press release, Armitage stated, “The primary advantages of our system are the simplicity of its design combined with the ease with which the fluorescence brightness and color can be tuned.”

According to Waggoner, this process also makes detection more sensitive so that the markers can contribute less volume to the structure. Furthermore, Waggoner said, “Since you can use many different kinds of dyes … you could perhaps get multiple reagents of different color so that you could detect several different things at once.”

Although this development marks an important advance in the field of DNA fluorescent marking, Armitage stated in the press release, “We really feel that this is the tip of the iceberg and that nanotags 100 times brighter than existing labels can be developed in any color.”