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A G-rich sequence was designed to allow folding into either a stable parallel or hybrid-type topology. With the parent sequence featuring coexisting species, various related sequences with single and double mutations and with a shortened central propeller loop affected the topological equilibrium. Two simple modifications, likewise introduced separately to all sequences, were employed to lock folds into one of the topologies without noticeable structural alterations. The unique combination of sequence mutations, high-resolution NMR structural information, and the thermodynamic stability for both topological competitors identified critical loop residue interactions. In contrast to first loop residues, which are mostly disordered and exposed to solvent in both propeller and lateral loops bridging a narrow groove, the last loop residue in a lateral three-nucleotide loop is engaged in stabilizing stacking interactions. The propensity of single-nucleotide loops to favor all-parallel topologies by enforcing a propeller-like conformation of an additional longer loop is shown to result from their preference in linking two outer tetrads of the same tetrad polarity. Taken together, the present studies contribute to a better structural and thermodynamic understanding of delicate loop interactions in genomic and artificially designed quadruplexes, e.g. when employed as therapeutics or in other biotechnological applications.
Abstract
G‐quadruplexes have attracted growing interest in recent years due to their occurrence in vivo and their possible biological functions. In addition to being promising targets for drug design, these four‐stranded nucleic acid structures have also been recognized as versatile tools for various technological applications. Whereas a large number of studies have yielded insight into their remarkable structural diversity, our current knowledge on G‐quadruplex stabilities as a function of sequence and environmental factors only gradually emerges with an expanding collection of thermodynamic data. This minireview provides an overview of general rules that may be used to better evaluate quadruplex thermodynamic stabilities but also discusses present challenges in predicting most stable folds for a given sequence and environment.
Abstract
Multiple G‐tracts within the promoter region of the c‐myc oncogene may fold into various G‐quadruplexes with the recruitment of different tracts and guanosine residues for the G‐core assembly. Thermodynamic profiles for the folding of wild‐type and representative truncated as well as mutated sequences were extracted by comprehensive DSC experiments. The unique G‐quadruplex involving consecutive G‐tracts II–V with formation of two one‐nucleotide and one central two‐nucleotide propeller loop, previously proposed to be the biologically most relevant species, was found to be the most stable fold in terms of its Gibbs free energy of formation at ambient temperatures. Its stability derives from its short propeller loops but also from the favorable type of loop residues. Whereas quadruplex folds with long propeller loops are significantly disfavored, a snap‐back loop structure formed by incorporating a 3’‐terminal guanosine into the empty position of a tetrad seems highly competitive based on its thermodynamic stability. However, its destabilization by extending the 3’‐terminus questions the significance of such a species under in vivo conditions.