Thursday, July 11, 2013

Applications Thursday: Molecular Beacon Helicase Assay

The Molecular Beacon Helicase Assay (MBHA) was designed to improve detection of helicase activity in order to identify small molecule inhibitors of helicase. The development of this assay is desirable due to the importance of helicases in maintaining infections mediated by viruses and other pathogens. The ability to maintain infections stems from the diverse functions of helicases in processes such as DNA replication and RNA transcription.

Simplified diagram of the structure of Hepatitis C virus
BMG LABTECH application note #241 describes an MBHA that was designed to detect inhibitors of the NS3 helicase from the hepatitis C virus. The performance of this MBHA relies on the molecular beacon substrate, which is two nucleotide strands annealed together. One of the strands has both a quencher and a fluorophore at either end so that when it is annealed to the other strand in the substrate a high level of fluorescence is detected. The strands of the substrate are further designed to form stem-loop structures once they have been separated such that the fluorophore and quencher are brought into proximity and fluorescence decreases when the strands are separated by an active helicase.
MBHA principle: Helicase (green) unwinds double-stranded
DNA using ATP. Resulting in fluorescent label (blue star)
that is brought close to a quencher (grey).

The experiments described were performed using a FLUOstar Omega but other BMG LABTECH microplate readers such as the POLARstar Omega,  PHERAstar FS and the CLARIOstar are compatible with this type of assay. For more information on this application and these products please visit: www.bmglabtech.com