The quest for a G-quadruplex specific fluorescent sensor among other DNA forms under physiological salt conditions has been addressed in this article. We demonstrate for the first time the application of a water-soluble fluorogenic dye, Thioflavin T (ThT), in a dual role of exclusively inducing quadruplex folding in the 22AG human telomeric DNA, both in the presence and absence of Tris buffer/salt, and sensing the same through its fluorescence light-up having emission enhancement of the order of 2100-fold in the visible region. Appropriate conditions allow an apparent switch over of the parallel quadruplex structure in 22AG-ThT (50 mM Tris, pH 7.2) solution to the antiparallel form just by the addition of K(+) ions in the range 10-50 mM. Moreover, addition of ThT cooperatively stabilizes the K(+) induced antiparallel quadruplexes by a ΔT(m) ∼11 °C. The distinction of ThT as a quadruplex inducer has been contrasted with the erstwhile used structurally related dye, Thiazole Orange (TO), which did not induce any quadruplex folding in the 22AG strand in the absence of salt. The striking fluorescence light-up in ThT on binding to the human telomeric G-quadruplex is shown to be highly specific compared to the less than 250-fold enhancement observed with other single/double strand DNA forms. This work has implication in designing new generation dyes based on the ThT scaffold, which are highly selective for telomeric DNA, for potential diagnostic, therapeutic, and ion-sensing applications.

Various biologically relevant G-quadruplex DNA structures offer a platform for therapeutic intervention for altering the gene expression or by halting the function of proteins associated with telomeres. One of the prominent strategies to explore the therapeutic potential of quadruplex DNA structures is by stabilizing them with small molecule ligands. Here we report the synthesis of bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine and their interaction with human telomeric DNA and promoter G-quadruplex forming DNAs. The interactions of ligands with quadruplex forming DNAs were studied by various biophysical, biochemical, and computational methods. Results indicated that bisquinolinium ligands bind tightly and selectively to quadruplex DNAs at low ligand concentration (∼0.2-0.4 μM). Furthermore, thermal melting studies revealed that ligands imparted higher stabilization for quadruplex DNA (an increase in the T(m) of up to 21 °C for human telomeric G-quadruplex DNA and >25 °C for promoter G-quadruplex DNAs) than duplex DNA (ΔT(m) ≤ 1.6 °C). Molecular dynamics simulations revealed that the end-stacking binding mode was favored for ligands with low binding free energy. Taken together, the results indicate that the naphthyridine-based ligands with quinolinium and pyridinium side chains form a promising class of quadruplex DNA stabilizing agents having high selectivity for quadruplex DNA structures over duplex DNA structures.

The linear syntheses of 4'-C-aminomethyl-2'-O-methyl uridine and cytidine nucleoside phosphoramidites were achieved using glucose as the starting material. The modified RNA building blocks were incorporated into small interfering RNAs (siRNAs) by employing solid phase RNA synthesis. Thermal melting studies showed that the modified siRNA duplexes exhibited slightly lower T(m) (∼1 °C/modification) compared to the unmodified duplex. Molecular dynamics simulations revealed that the 4'-C-aminomethyl-2'-O-methyl modified nucleotides adopt South-type conformation in a siRNA duplex, thereby altering the stacking and hydrogen-bonding interactions. These modified siRNAs were also evaluated for their gene silencing efficiency in HeLa cells using a luciferase-based reporter assay. The results indicate that the modifications are well tolerated in various positions of the passenger strand and at the 3' end of the guide strand but are less tolerated in the seed region of the guide strand. The modified siRNAs exhibited prolonged stability in human serum compared to unmodified siRNA. This work has implications for the use of 4'-C-aminomethyl-2'-O-methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs.

Various potential G-quadruplex forming sequences present in the genome offer a platform to modulate their function by means of stabilizing molecules. Though G-quadruplex structures exhibit diverse structural topologies, the presence of G-quartets as a common structural element makes the design of topology specific ligands a daunting task. To address this, the subtle structural variations of loops and grooves present in the quadruplex structures can be exploited. To this end, we report the design and synthesis of quadruplex stabilizing agents based on bisbenzimidazole carboxamide derivatives of pyridine, 1,8-naphthyridine, and 1,10-phenanthroline. The designed ligands specifically bind to and stabilize promoter quadruplexes having parallel topology over any of the human telomeric quadruplex topologies (parallel, hybrid, or antiparallel) and duplex DNAs. CD melting studies indicate that ligands could impart higher stabilization to c-MYC and c-KIT promoter quadruplexes (up to 21 °C increment in Tm) than telomeric and duplex DNAs (ΔTm ≤ 2.5 °C). Consistent with a CD melting study, ligands bind strongly (Kb = ∼10(4) to 10(5) M(-1)) to c-MYC quadruplex DNA. Molecular modeling and dynamics studies provide insight into how the specificity is achieved and underscore the importance of flexible N-alkyl side chains attached to the benzimidazole-scaffold in recognizing propeller loops of promoter quadruplexes. Overall, the results reported here demonstrate that the benzimidazole scaffold represents a potent and powerful side chain, which could judiciously be assembled with a suitable central core to achieve specific binding to a particular quadruplex topology.

The formation of G-quadruplex structures can regulate telomerase activity and the expression of oncogenes at the transcriptional and translational levels. Therefore, stabilization of G-quadruplex DNA structures by small molecules has been recognized as a promising strategy for anticancer drug therapy. One of the major challenges in this field is to impart stabilizing molecules with selectivity toward quadruplex structures over duplex DNAs, and to maintain specificity toward a particular quadruplex topology. Herein we report the synthesis and binding interactions of indenopyrimidine derivatives, endowed with drug-like properties, with oncogenic promoters of c-myc and c-kit, telomeric and duplex DNAs. The results show specific stabilization of promoter over telomeric quadruplexes and duplex DNAs. Molecular modeling studies support the experimental observations by unraveling the dual binding mode of ligands by exploiting the top and bottom quartets of a G-quadruplex structure. This study underscores the potential of the indenopyrimidine scaffold, which can be used to achieve specific G-quadruplex-mediated anticancer activity.