A novel strategy has recently been developed which allows the visualization of DNA synthesis in intact cells or organisms. This strategy, developed by the University of Zurich’s Institute of Organic Chemistry, utilizes a tailor-made nucleic acid which can be incorporated into a normal DNA strand to allow this biochemical process to be seen using fluorescent probes.

While this is not the first time that synthetic nucleotides have been utilized for DNA labeling in biochemical assays, it does appear to be the first successful utilization of a non-toxic strategy for doing so. Previous attempts to accomplish this feat have been characterized by significant toxic consequences for the cells or tissues being analyzed, making these approaches inappropriate for both in-vivo analyses and assays in which the integrity of native metabolic pathways must be preserved.

Collectively, these types of strategies are known as bioorthogonal chemical reporter assays, and involve the synthetic addition of a specific functional group (aldehydes, ketones, certain amino acids, etc.) to a naturally occurring biomolecule such as DNA. These additions are often made through the manipulation of native metabolic processes, and allow for subsequent “tagging” and visualization of the attached biomolecule. In the present study, the researchers utilized a specially designed nucleoside (the monomeric units which DNA is composed of) containing a simple alkyne functional group to create this addition. Once these nucleosides are incorporated into a DNA molecule, the alkyne (carbon-carbon triple bond) functional group can be easily coupled to a fluorescent tag to allow visualization.

This type of DNA labeling has traditionally been accomplished through the use of similar synthetic nucleosides, including BrdU and EdU. The former contains a bromine atom in place of the alkyne functional group of the present study, and though these differences seem minimal, BrdU can act as a potent mutagen when utilized in these labeling assays. Additionally, the processing of samples requires both DNA denaturation and fixation, both of which serve to kill the cells which contain this DNA. More recent studies which have utilized EdU show that, though this nucleoside allows for more sensitive visualization, this advantage is outweighed by the fact that the compound causes cell-cycle arrest and that its toxicity rivals that of BrdU. Depending on how these compounds are utilized, a combination of antibody staining and autoradiography can also make these strategies incompatible with goals that require in vivo DNA visualization.

The newly designed nucleoside, known as F-ara-Edu, was developed by Anne Neef, a graduate student at the University of Zurich. This construct replaces the normal nucleoside thymidine, and causes virtually no issues with regard to genome stability or cell cycle progression. Though there could be enormous potential for the utilization of this new strategy in a variety of contexts, two ideas were specifically addressed by Neef’s research team; these include the identification of sites in which either cancer growth or viral infections are present. In both cases, levels of DNA synthesis should be much greater than that seen in normal surrounding cells, allowing rapid identification of potentially affected regions or groups of cells.

Preliminary studies in zebrafish have already shown that, by injecting an organism with F-ara-Edu while it is still at the single-cell embryonic stage, cells undergoing differentiation at a later developmental stage can be identified as the organism develops over time. It will be interesting to see how this new visualization technique affects future strategies utilized in both drug research and biotechnology.

Original Research Paper: http://www.pnas.org/content/early/2011/11/28/1101126108

Original Press Release: http://www.mediadesk.uzh.ch/articles/2011/dns-synthese-im-lebenden-organ...
*Photo taken from the original University of Zurich press release