Characterization of G-Quadruplex Motifs in espB, espK, and cyp51 Genes of Mycobacterium tuberculosis as Potential Drug Targets

Mishra, Subodh Kumar; Shankar, Uma; Jain, Neha; Sikri, Kriti; Tyagi, Jaya Sivaswami; Sharma, Tarun Kumar; Mergny, Jean-Louis; Kumar, Amit

However, the Taq polymerase stop assay assesses the ability of TMPyP4 to stabilize the G4 structure and stop the movement of polymerase enzyme during PCR extension reaction at MTB-PGQ sites. 46 Figure S8 depicts the results of Taq polymerase stop assay for the three templates and negative control. In the absence of TMPyP4, full-length product is formed but with increasing concentrations of TMPyP4, the shorter band was observed with the decreasing band intensity, whereas neither smaller product nor diminishing bands intensities were observed in the negative control. 46 TMPyP4 Reduced the Transcription of espK, espB, and cyp51 Genes Next, we sought to examine the effect of TMPyP4 on transcription of the G4 motif-possessing genes espK, espB, and cyp51 by qRT-PCR. The transcripts of genes were quantified relative to the transcript of 16S rRNA gene (a housekeeping gene), and quantification cycle (Cq) of non-template controls was >35 cycles. As shown in Figure 6 , treatment of the M. tuberculosis cells with 6.25 mM TMPyP4 reduced the transcription of the espK, espB, and cyp51 genes by 3.70-, 1.91-, and 6.25-fold, respectively, relative to the untreated culture control. These results strengthen the likelihood of a role for a G4-mediated mechanism in the transcription of the G4 motif-containing genes espK, espB, and cyp51, and open a novel therapeutic avenue for combating this deadly human pathogen.