Interactions of Actinomycin D with Human Telomeric G-Quadruplex DNA (original) (raw)

The G-quadruplex structural motif of DNA has emerged as a novel and exciting target for anticancer drug discovery. The human telomeric G-quadruplex consists of a single strand repeat of d[AGGG(TTAGGG) 3 ] that can fold into higher-order DNA structures. Small molecules that selectively target and stabilize the G-quadruplex structure(s) may serve as potential therapeutic agents and have garnered significant interest in recent years. In the work presented here, the anticancer agent, actinomycin D, is demonstrated to bind to and induce changes in both structure and stability to both the Na + and K + forms of the G-quadruplex DNA. The binding of actinomycin D to the G-quadruplex DNAs are characterized by intrinsic association constants of approximately 2 × 10 5 M −1 (strand), 2:1 molecularity, and are shown to be enthalpically driven with binding enthalpies of approximately −7 kcal/mol. The free Na + or K + forms of the quadruplex structures differ in melting temperatures by approximately 8°C (60 and 68°C, respectively), whereas both forms, when complexed with actinomycin D are stabilized with melting temperatures of approximately 79°C. The induced CD signals observed for the actinomycin D-G-quadruplex complexes may indicate that the phenoxazone ring of actinomycin D to be stacked on the G-tetrad rather than intercalated between adjacent G-tetrads. Complex formation with actinomycin D results in changes to both the Na + or K + structural isoforms to "ligand-bound" complexes having similar structural properties and stabilities. The DNA structural motif known as the G-quadruplex has recently emerged as a novel and exciting target for the discovery and design of new classes of anticancer agents (1-3). DNA sequences that can form G-quadruplex structures are found extensively throughout the genome and are located in biologically relevant regions. These sequences and their corresponding quadruplex structures were first observed to exist in telomeric regions of DNA, located at the terminal ends of chromosomes (4-6). More recently, G-quadruplex forming sequences have been mapped to the promoter regions of a number of genes and oncogenes (7-9). It is postulated that G-quadruplex structures may serve important biological functions in the regulation of gene expression (10-12). Hence, these findings have led to an increased interest in the structural and functional features of G-quadruplex structural motif and offers potentially novel targets for the development of small molecules that could selectively target and stabilize the quadruplex structure. The G-quadruplex consists of stacked G-tetrads connected by lateral, diagonal, or external loops and has been shown to possess a wide range of structural polymorphism within guanine rich sequences that exhibit the motif. Burge and coworkers and Dai and coworkers recently published extensive reviews of the topologies of quadruplex DNA structures (13,14). The observed polymorphism(s) found for G-quadruplex structures include differences in molecularity, strand orientation, loop characteristics, and structural isomers based on selected cation buffer conditions. The structural stability of quadruplex DNAs have

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