Abstract
G-quadruplexes have been characterized as structures of vital importance in the cellular functioning of several life forms. They have subsequently been established to serve as a therapeutic target of several diseases including cancer, HIV, tuberculosis and malaria. In this paper, we report the binding of aminosugar-intercalator conjugates with a well-studied anti-parallel G-quadruplex derived from Oxytricha Nova G-quadruplex DNA. Of the four neomycin-intercalator conjugates studied with varying surface areas, BQQ-neomycin conjugate displayed the best binding to this DNA G-quadruplex structure with an association constant of Ka = (1.01 ±0.03) × 107 M−1 which is nearly 100-fold higher than the binding of neomycin to this quadruplex. The binding of BQQ-neomycin displays a binding stoichiometry of 1:1 indicating the presence of a single and unique binding site for this G-quadruplex. In contrast, the BQQ-neomycin displays very weak binding to the bacterial A-site rRNA sequence showing that BQQ-does not enhance the neomycin binding to its natural target, the bacterial rRNA A-site. The BQQ-neomycin conjugate is prone to aggregation even at low micromolar concentrations (4 μM) leading to some ambiguities in the analysis of thermal denaturation profiles. Circular dichroism experiments showed that binding of BQQ-neomycin conjugate causes some structural changes in the quadruplex while still maintaining the overall anti-parallel structure. Finally, the molecular docking experiments suggest that molecular surface plays an important role in the recognition of a second site on the G-quadruplex. Overall, these results show that molecules with more than one binding moieties can be made to specifically recognize G-quadruplexes with high affinities. The dual binding molecules comprise of quadruplex groove binding and intercalator units, and the molecular surface of the intercalator plays an important part in enhancing binding interaction to the G-quadruplex structure.
Highlights
G-quadruplex structures are highly polymorphic DNA structures that can exist in dynamic equilibrium with other possible orientations of the same structure (Seenisamy et al, 2004)
This assay exploits the fluorescence enhancement of nucleic acid binding dyes that show non-specific binding and moderate affinities to their cognate structures. The intercalating dyes such as ethidium bromide or Thiazole orange are traditional intercalators used in this assay but recently the use of dyes such as Thioflavin T (ThT) is gaining momentum due to its special liking for certain G-quadruplex structures (Mohanty et al, 2012; Verma et al, 2019)
The ratio of nucleic acid to the intercalator to be used in this assay is determined usually by running a fluorescence titration experiment in which the nucleic acid of interest is titrated against the intercalator ligand and the resulting fluorescence changes are used to construct a plot giving the stoichiometry of the intercalatornucleic acid interaction
Summary
G-quadruplex structures are highly polymorphic DNA structures that can exist in dynamic equilibrium with other possible orientations of the same structure (Seenisamy et al, 2004). Several other life forms such as bacteria, fungus and viruses have been shown to possess regions capable of forming G-quadruplex and they have been suggested to play prominent roles in their life progression (Thakur et al, 2014; Artusi et al, 2015; Perrone et al, 2017). Due to these advances in the field, G-quadruplexes have moved far away from being a cancer-centric target to a structure which has relevance in the design of new antibacterial, antifungal and antiviral agents
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