Quadruplex Nucleic Acids

Structural studies have shown that four G-tracts along a DNA strand are the minimal requirement for intramolecular G-quadruplex formation. Longer DNA sequences containing multiples of four G-tracts could, in principle, form higher-order structures based on multiple G-quadruplex blocks. This latter condition is abundantly verified for the telomeric single-stranded overhang (~200 nt) consisting of tens of TTAGGG repeats, thus opening new interesting questions about the structure of the “real” telomeric DNA. How many quadruplex units form in the human telomeric overhang? Which type of quadruplex topologies? Do they interact or not? What about their binding properties? Although many of these questions are still unanswered, recent experimental and computational studies have begun to address them. The existence and relevance of these higher-order quadruplex structures in the human genome is now an interesting and stimulating research topic in the quadruplex field. The recent results, the unsolved problems, and the future prospects for understanding higher-order telomeric quadruplex structures are the main topics of this review. Other studies on long telomeric RNA sequences and on other intramolecular (non telomeric) DNA higher order quadruplex structures are also presented.

In this chapter we describe the application of in-cell NMR spectroscopy to the investigation of G-quadruplex structures inside living Xenopus laevis oocytes and in X. laevis egg extract. First, in-cell NMR spectroscopy of nucleic acids (NA) is introduced and applications and limitations of the approach are discussed. In the following text the application of in-cell NMR spectroscopy to investigation of G-quadruplexes are reviewed. Special emphasis is given to the discussion of the influence of the intracellular environmental factors such as low molecular weight compounds, molecular crowding, and hydration on structural behavior of G-quadruplexes. Finally, future perspectives of in-cell NMR spectroscopy for quantitative characterization of G-quadruplexes and NA are discussed.

Intracellular space is highly crowded with soluble and insoluble biomolecules that range from large polymers, such as proteins and nucleic acids, to small molecules, including metabolites and metal ions. It is therefore of interest to understand the effects of molecular crowding on the structure, stability, and function of biomolecules. Moreover, molecular crowding is observed not only intracellularly but also in the extracellular matrix and under the conditions used in in vitro biotechnological and nanotechnological processes. However, most biochemical studies of biomolecules are performed under dilute conditions. Recent studies have demonstrated critical effects of molecular crowding on nucleic acids. In the present study we discuss how molecular crowding affects the properties of G-quadruplexes as well as other non-canonical nucleic acid structures.

G-quadruplexes under molecular crowding conditions

G-quadruplex ligands are potential anticancer agents as telomerase inhibitors and potential transcriptional regulators of oncogenes. The search for best-in-class drugs is addressed to identify small molecules able to promote and stabilize G-quadruplex structures. What features should the G-quadruplex ligands possess? They should have selective antiproliferative effects on cancer cells and induce telomerase inhibition or oncogene suppression. One of the main challenges in their design and synthesis is to make the ligands selective for G-quadruplex DNA. These features should be amplified by careful analyses of physico-chemical aspects of G-quadruplex-drug interactions. In particular, the study of the energetics of G-quadruplex-drug interactions can enhance drug design by providing thermodynamic parameters that give quantitative information on the biomolecular interactions important for binding. The main methodologies used to gain information on energetics of binding are based on spectroscopic or calorimetric principles. Spectroscopic techniques such as fluorescence and circular dichroism are rapid and cheap methods, but are not sufficient to characterize completely the thermodynamics of interaction. Calorimetric techniques such as isothermal titration calorimetry offer a direct measure of binding enthalpy, in addition to the stoichiometry and affinity constants. With the complete thermodynamic signature of drug-target interaction, dissecting the enthalpic and entropic components of binding is possible, which can be a useful aid to decision-making during drug optimization.