Nucleic Acid Cleavage Assay for Transposase/Integrase Protein Superfamily
Topics in This Section
Tn5 transposase and HIV integrase are members of the transposase/integrase protein superfamily. Although their primary sequences are very different, they share extensive functional and 3-D structural identity, suggesting that agents that alter the activity of Tn5 transposase may also affect HIV integrase. Such agents may be potential therapeutics for HIV infection.
Dr. Anna Skalka in collaboration with UW-Madison researchers have developed a fluorescence polarization assay for monitoring the nucleic acid cleavage activity of a member of the transposase/integrase superfamily, such as Tn5 transposase. For the assay, a target DNA molecule is fluorescently labeled. Large molecules have greater levels of fluorescence polarization than small molecules. When a transposase or integrase binds to the labeled DNA, creating a large complex, the fluorescence polarization increases. Cleavage of the DNA can be detected by observing the decrease in fluorescence polarization that occurs when the DNA molecule is cleaved into smaller fragments. Compounds that inhibit or enhance nucleic acid cleavage can thus be identified by their effect on fluorescence polarization.
- Identifying compounds that modulate the nucleic acid cleavage activity of a member of the transposase/integrase superfamily
- Compounds identified using this method may be useful for preventing and treating HIV infection
- Easily adapted for high throughput applications
- Involves no washing step
- Requires minimal sample volume
Ason B., Knauss D.J., Balke A.M., Merkel G., Skalka A.M. and Reznikoff W.S. Targeting Tn5 Transposase Identifies Human Immunodeficiency Virus Type 1 Inhibitors. Antimicrob. Agents. Chemother. (2005); 49, 2035-2043.
U.S. patent 2009/0098548: Modulators of Enzymatic Nucleic Acid Elements Mobilization