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

Circular dichroism is among the most dependable methods to portray the formation of G4 structures by DNA oligonucleotides. Previous studies have observed that: (1) a positive peak at $265 nm and a negative peak at $240 nm correspond to a parallel G4, whereas (2) a positive peak at 290 nm and a negative peak at 260 nm indicate antiparallel G4 formation, and (3) two positive peaks at 260 and 290 nm coupled with a negative peak at 240 nm represent a mixed or hybrid G4 structure. 34 Different cations exert variable effects on the stability and folding pattern of G4 structures. The ranking of stabilizing ability among the well-studied cations is as follows: K + > Na + > Mg 2+ > Li + . 35 Therefore, we assessed the topology of conserved MTB-PGQs using CD spectra and CD melting curve analysis in four different buffers containing related cations K + , Na + , Mg + , and Li + . CD spectra analysis revealed a predominantly parallel G4 topology exhibited by all conserved MTB-PGQs. As expected, the molar ellipticity and melting temperature of each MTB-PGQ were found to be higher in the presence of K + ions (Figure 2 ). CD spectra analysis performed in the presence of an increasing concentration of K + ions revealed an increase in molar ellipticity as a function of K + ion concentration ( Figure S5 ). We then mutated the central guanine nucleotide of all of the G stretches into thymine and evaluated the G4-forming ability of mutated MTB-PGQs (mut-MTB-PGQs) in K + buffer (Table S10) . All of the mutants (mut-MTB-PGQ1, mut-MTB-PGQ2, and mut-MTB-PGQ3) did not show CD signals that correspond to G4 topology and suggested the disruption of the G4 structure formation by the guanine mutation (Figure 2 ).