Mycobacterium tuberculosis (Mtb) contains potential G-quadruplex (PGQ) motifs in the genes espK and cyp51, which are crucial for the bacteria's virulence within host cells. Aminoglycoside molecules are commonly used as antibiotics for ribosomal targets. This study provides insight into the interactions between these aminoglycosides and Mtb-PGQ sequences (espK and cyp51), shedding light on the structural and thermodynamic dynamics of their binding. This study demonstrates the stability, affinity, and conformation of Mtb-PGQ in the presence of neomycin and streptomycin. Ultraviolet-visible spectroscopy (UV-vis), circular dichroism spectroscopy (CD), CD thermal melting, isothermal titration calorimetry (ITC), and fluorescence intercalator displacement (FID) assays were used to comprehensively examine these interactions. Our results reveal that neomycin with Mtb-PGQexhibits hypochromism accompanied by a 4-5 nm red shift in the UV-visible absorption titration, whereas streptomycin exhibits a hypochromic shift without changes in the maximum wavelength. Notably, neomycin shows a nonlinear binding isotherm, suggesting the involvement of more than one binding process in the formation of neomycin.Mtb-PGQ complexes. Scatchard plot analysis indicates higher binding affinity values for neomycin compared with weaker affinity of streptomycin. CD studies reveal that neomycin decreases the ellipticity of Mtb-PGQ with a red shift while retaining the parallel topology, ultimately enhancing the thermal stability of both espK and cyp51. In contrast, streptomycin destabilizes the cells. ITC analysis reveals that neomycin exhibits the strongest binding affinity for cyp51, with the relative order being NEO-cyp51 > NEO-espk > STR-cyp51 > STR-espk. Moreover, thermodynamic analysis reveals that neomycin possesses a unique dual mode of binding through grooves as well as stacking. FID studies further confirm a lower DC50 value for neomycin than for streptomycin, suggesting that neomycin is a strong displacer of thiazole orange. Thus, the results show that neomycin with amino groups selectively recognizes the grooves of cyp51 over espK.
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