Ligand-Induced Dimerization of a Truncated Parallel MYC G-Quadruplex.

Binding of an indoloquinoline derivative with an aminoalkyl side chain to a truncated sequence from the MYC promoter region was studied through isothermal titration calorimetry (ITC). The targeted MYC3 sequence lacks 3'-flanking nucleotides and forms a monomeric parallel quadruplex (G4) with a blunt-ended 3'-outer tetrad under the solution conditions employed. Analysis of ITC isotherms reveals multiple binding equilibria with the initial formation of a 1:2 ligand/quadruplex complex. Evaluation of electrophoretic mobilities as well as NMR spectral data confirm ligand-induced dimerization of MYC3 quadruplexes with the ligand sandwiched between the two 3'-outer tetrads. Additional ligand molecules in excess bind to the 5'-outer tetrads of the sandwich complex. Such a ligand-promoted G4 dimerization may be exploited for the controlled assembly or disassembly of G4 aggregates to expand on present quadruplex-based technologies.

Observation of a Dynamic G-Tetrad Flip in Intramolecular G-Quadruplexes.

A MYC sequence forming an intramolecular G-quadruplex with a parallel topology was modified by the incorporation of 8-bromoguanosine (BrG) analogues in one of its outer G-tetrads. The propensity of the BrG analogues to adopt a syn glycosidic torsion angle results in an exceptional monomolecular quadruplex conformation featuring a complete flip of one tetrad while keeping a parallel orientation of all G-tracts as shown by circular dichroism and nuclear magnetic resonance spectroscopic studies. When substituting three of the four G-tetrad residues with BrG analogues, two coexisting quadruplex conformational isomers with an all-syn and all-anti outer G-quartet are approximately equally populated in solution. A dynamic interconversion of the two quadruplexes with an exchange rate (kex) of 0.2 s-1 is demonstrated through the observation of exchange crosspeaks in rotating frame Overhauser effect spectroscopy and nuclear Overhauser effect spectroscopy experiments at 50 °C. The kinetic properties suggest disruption of the corresponding outer G-tetrad but not of the whole quadruplex core during the tetrad flip. Conformational syn-anti isomers with homopolar and heteropolar stacking interactions are nearly isoenergetic with a transition enthalpy of 18.2 kJ/mol in favor of the all-syn isomer.

Towards the Development of Structure-Selective G-Quadruplex-Binding Indolo[3,2-b]quinolines.

The interaction of phenyl-substituted indolo[3,2-b]quinolines with DNA G-quadruplexes of different topology were studied by using a combination of spectroscopic and calorimetric methodologies. N5-Methylated indoloquinoline derivatives ((Me) PIQ) with an aminoalkyl side chain exhibit high affinities for the parallel-stranded MYC quadruplex and a (3+1)-hybrid structure combined with an excellent discrimination against the antiparallel thrombin-binding aptamer (TBA) and the human telomeric (HT) quadruplexes. Dissociation constants for the binding of the ligand to the MYC quadruplex are in the submicromolar range, being below the corresponding dissociation constants for the antiparallel-stranded quadruplexes by about one order of magnitude. Competition experiments with double-helical DNA reveal the impact of indoloquinoline structural features on the selectivity for the parallel quadruplex relative to duplex DNA. Based on a calorimetric analysis binding to MYC is shown to be equally driven by favorable enthalpic and entropic contributions with no significant impact on the type of cation present.

Molecular Recognition and Visual Detection of G-Quadruplexes by a Dicarbocyanine Dye.

The interactions of a dicarbocyanine dye 3,3'-diethylthiadicarbocyanine, DiSC2(5), with DNA G-quadruplexes were studied by means of a combination of various spectroscopic techniques. Aggregation of excess dye as a result of its positive charge is promoted by the presence of the polyanionic quadruplex structure. Specific high-affinity binding to the parallel quadruplex of the MYC promoter sequence involves stacking of DiSC2(5) on the external G-tetrads; the 5'-terminal tetrad is the favored binding site. Significant energy transfer between DNA and the dye in the UV spectral region is observed upon DiSC2(5) binding. The transfer efficiency strongly depends on the DNA secondary structure as well as on the G-quadruplex topology. These photophysical features enable the selective detection of DNA quadruplexes through sensitized DiSC2(5) fluorescence in the visible region.

Revealing the Energetics of Ligand-Quadruplex Interactions Using Isothermal Titration Calorimetry.

The thermodynamic characterization of G4-ligand interactions has shown to be a powerful adjunct to structural information in the rational design and optimization of potent G-quadruplex ligands for use in therapeutics, diagnostics, or other technological applications. Isothermal titration calorimetry (ITC) can resolve energetic contributions to complex formation and constitutes the only available experimental method to directly measure binding enthalpies. A general protocol for using ITC in studies on quadruplex-ligand interactions with details on the experimental setup, data analysis, and potential pitfalls is presented. The methodologies used are illustrated on results obtained from the targeting of a parallel DNA G-quadruplex with a G4-binding indoloquinoline derivative.

Thermodynamic signature of indoloquinolines interacting with G-quadruplexes: Impact of ligand side chain.

Binding of indoloquinolines with different aliphatic side chains to a parallel G-quadruplex derived from the MYC promoter sequence was characterized by optical and calorimetric measurements. ITC experiments performed at different temperatures enabled the determination of molar heat capacity changes upon quadruplex binding and a partitioning of the total binding free enthalpy into contributing terms with hydrophobic effects being major driving forces for all derivatives. Whereas affinities increase for indoloquinolines equipped with a long and positively charged side arm, the highest contribution of specific intermolecular interactions anticipated to impart enhanced specificity is found for a ligand with an uncharged ether aliphatic tail. Obtained thermodynamic signatures may considerably aid in the rational selection of ligand side chains for G-quadruplex binders with enhanced affinity or selectivity.

Comprehensive Thermodynamic Profiling for the Binding of a G-Quadruplex Selective Indoloquinoline.

Binding of a positively charged indoloquinoline derivative to a G-quadruplex formed by the G-rich promoter element of the c-MYC oncogene was subjected to a rigorous isothermal calorimetric analysis. Binding of the indoloquinoline is primarily enthalpy-driven but is also promoted by a favorable entropy term. Both binding enthalpy ΔH° and binding entropy ΔS° exhibit a noticeable temperature dependence with almost complete enthalpy-entropy compensation as a result of a negative change in heat capacity ΔCp°. Salt-dependent polyelectrolyte effects only moderately contribute to the overall free energy of association. More details on the binding process are revealed in an attempt to dissect the total free energy into individual contributory terms. Accordingly, specific intermolecular interactions between the indoloquinoline ligand and G-quadruplex substantially contribute in addition to hydrophobic effects in promoting the association. Comparing thermodynamic profiles for various quadruplex ligands indicates different energetic patterns that may aid in the rational design of more efficient quadruplex binding ligands in the future.