A Short Interfering RNA (siRNA) Molecular Beacon for the Detection of Mycobacterial Infection

Abstract

In latent TB, the ability of Mycobacterium tuberculosis to invade and survive within macrophages of the pulmonary granuloma is attributed to protein products of mammalian cell entry (mce4) operon genes (A-F). These are cholesterol transporters which facilitate the transport of host lipids into the mycobacterium allowing long term survival during chronic infection. Currently, there are no rapid and reliable tests for the detection of latent TB. Therefore, because there is a lack of reliable and efficient tests for the diagnosis of latent TB, we tested the hypothesis that mycobacterial infection can be detected using mce4 siRNA molecular beacons against mce4 mRNAs. Because our work showed that the mce4A gene of the mce4 operon conferred infectivity to host E. coli, a siRNA molecular beacon was designed against a region of the mce4A mRNA that is highly homologous in Mycobacterium tuberculosis and Mycobacterium smegmatis. This molecular beacon has a hairpin structure with a stem, 5 nucleotides on either end that are complementary to each other, and a loop which contains 20 nucleotides that are complementary to a region of the target mRNA. Conjugated to the 5’ and 3’ ends of the molecular beacon are the fluorophore TYE 665 and quencher Iowa Black RQ-SP. In the absence of the target mRNA the hairpin structure will predominate and fluorescence will be quenched while in the presence of the target mRNA fluorescence will be induced. Our study shows that the siRNA molecular beacon detects its target in M. smegmatis and in macrophages infected with M. smegmatis and offers a potential test for detection of mycobacterial infection.