The effect of selective REM-sleep deprivation on the consolidation and affective evaluation of emotional memories.

Emotion boosts the consolidation of events in the declarative memory system. Rapid eye movement (REM) sleep is believed to foster the memory consolidation of emotional events. On the other hand, REM sleep is assumed to reduce the emotional tone of the memory. Here, we investigated the effect of selective REM-sleep deprivation, SWS deprivation, or wake on the affective evaluation and consolidation of emotional and neutral pictures. Prior to an 9-h retention interval, sixty-two healthy participants (23.5 ± 2.5 years, 32 female, 30 male) learned and rated their affect to 80 neutral and 80 emotionally negative pictures. Despite rigorous deprivation of REM sleep or SWS, the residual sleep fostered the consolidation of neutral and negative pictures. Furthermore, emotional arousal helped to memorize the pictures. The better consolidation of negative pictures compared to neutral ones was most pronounced in the SWS-deprived group where a normal amount of REM sleep was present. This emotional memory bias correlated with REM sleep only in the SWS-deprived group. Furthermore, emotional arousal to the pictures decreased over time, but neither sleep nor wake had any differential effect. Neither the comparison of the affective ratings (arousal, valence) during encoding and recognition, nor the affective ratings of the recognized targets and rejected distractors supported the hypothesis that REM sleep dampens the emotional reaction to remembered stimuli. The data suggest that REM sleep fosters the consolidation of emotional memories but has no effect on the affective evaluation of the remembered contents.

Exploring multiple binding sites of an indoloquinoline in triple-helical DNA: a paradigm for DNA triplex-selective intercalators.

Employing NMR spectroscopic methods preferred binding sites of a triplex-selective indoloquinoline drug were examined with three DNA triplex targets. To directly derive and evaluate number and type of the different sites of interaction, studies were performed on short triple-helical constructs specifically labeled with 3-(15)N thymidine probes. The detection and assignment of several coexisting species was enabled through the observation of slow exchange on the chemical shift timescale between complexes and free triplex. In general, the 5'-triplex-duplex junction constitutes the most favorable intercalation site, in particular when flanked by a TAT base triad. NMR data also revealed two different orientations for the intercalating indoloquinoline drug. Binding affinity significantly decreases with a C(+)GC triad bordering the junction but junction binding is still preferred over intercalation between TAT base triads within the triplex stem. In addition to the intercalation between two uncharged TAT triplets, intercalation between a TAT and a 3'-terminal C(+)GC triplet was also observed, indicating a non-protonated third strand cytosine at the triplex end position.

Spectroscopic and calorimetric studies on the binding of an indoloquinoline drug to parallel and antiparallel DNA triplexes.

11-Phenyl-substituted indoloquinolines have been found to exhibit significant antiproliferative potency in cancer cells but to show only moderate affinity toward genomic double-helical DNA. In this study, parallel as well as antiparallel triple-helical DNA targets are employed to evaluate the triplex binding of these ligands. UV melting experiments with parallel triplexes indicate considerable interactions with the drug and a strong preference for TAT-rich triplexes in line with an increasing number of potential intercalation sites of similar binding strength between two TAT base triads. Via substitution of a singly charged aminoethylamine side chain by a longer and doubly charged bis(aminopropyl)amine substituent at the ligand, binding affinities increase and also start to exhibit long-range effects as indicated by a strong correlation between the binding affinity and the overall length of the TAT tract within the triplex stem. Compared to parallel triplexes, an antiparallel triplex with a GT-containing third strand constitutes a preferred target for the indoloquinoline drug. On the basis of pH-dependent titration experiments and corroborated by a Job analysis of continuous variation, binding of the drug to the GT triplex not only is strongly enhanced when the solution pH is lowered from 7 to 5 but also reveals a pH-dependent stoichiometry upon formation of the complex. Calorimetric data demonstrate that stronger binding of a protonated drug at acidic pH is associated with a more exothermic binding process. However, at pH 7 and 5, binding is enthalpically driven with additional favorable entropic contributions.

Binding and NMR structural studies on indoloquinoline-oligonucleotide conjugates targeting duplex DNA.

An 11-phenyl-indolo[3,2-b]quinoline (PIQ) was tethered through an aminoalkyl linker to the 5'-end of four pyrimidine oligonucleotides with T/C scrambled sequences at their two 5'-terminal positions. Binding to different double-helical DNA targets formed parallel triple helices with a PIQ-mediated stabilization that strongly depends on pH and the terminal base triad at the 5'-triplex-duplex junction. The most effective stabilization was observed with a TAT triplet at the 5'-junction under low pH conditions, pointing to a protonated ligand with a high triplex binding affinity and unfavorable charge repulsions in the case of a terminal C(+)GC triplet at the junction. The latter preference of the PIQ ligand for TAT over CGC is alleviated yet still preserved at higher pH. Intercalation of PIQ at the 5'-triplex-duplex junction as suggested by the triplex melting experiments was confirmed by homonuclear and heteronuclear NMR structural studies on a specifically isotope-labeled triplex. The NMR analysis revealed two coexisting species that only differ by a 180° rotation of the indoloquinoline within the intercalation pocket. NOE-derived molecular models indicate extensive stacking interactions of the indoloquinoline moiety with the TAT base triplet and CG base pair at the junction and a phenyl substituent that is positioned in the major groove and oriented almost perpendicular to the plane of the indoloquinoline.

Low-temperature NMR studies on inosine wobble base pairs.

Base pairs formed by the inosine nucleoside (I) play an important role in many physiological processes as well as in various DNA technologies. Relative stabilities and favored base pair geometries of free inosine wobble base pairs in aprotic solvents have been determined through (1)H NMR measurements at room temperature and at very low temperatures in a freonic solvent. As indicated by its significantly deshielded imino proton, the Watson-Crick-type I·C base pair forms a remarkably strong NHN hydrogen bond. For the thermodynamically less stable I·A wobble base pair, two configurations of similar population coexist at 133 K in the slow hydrogen bond exchange regime, namely a Watson-Crick(I)-Watson-Crick(A) geometry and a Watson-Crick(I)-Hoogsteen(A) geometry. I·U base pairs are stabilized by two rather weak hydrogen bonds and are significantly disfavored over inosine self-associates in a low-temperature Freon solution.

In vitro anticancer activity and evaluation of DNA duplex binding affinity of phenyl-substituted indoloquinolines.

Phenyl-substituted indoloquinolines were studied for their biological activity and their DNA binding affinity. Water-soluble aminoalkyl derivatives were prepared and have shown significant in vitro anticancer activity. Unlike previous reports on the potential role of duplex DNA as target for various indoloquinoline based drugs, duplex UV melting experiments and fluorescence titrations suggest only weak and moderately strong binding of the phenyl-substituted indoloquinolines at 120 mM and 20 mM Na(+) concentrations, respectively. Binding is suggested by ethidium displacement and circular dichroism experiments to be associated with drug intercalation between base pairs.

Spectroscopic and calorimetric studies on the triplex formation with oligonucleotide-ligand conjugates.

Several triplex-forming 9-mer oligonucleotide (TFO) conjugates with a methyl- or methoxy-substituted 5-phenyl-6H-indolo[3,2-b]quinoline (PIQ) attached at the 5'-terminus or 3'-terminus or at an internal C5 thymine position were synthesized and tested for their ability to form and stabilize a triple helix with a double-helical DNA target employing UV melting experiments, fluorescence titrations, and isothermal titration calorimetry (ITC). A considerable thermal stabilization by up to 14 degrees C at pH 6.0 was observed for the 5'- and 3'-conjugates with little influence on the type of substituent but also for a conjugate with the ligand tethered by a short linker to the interior of the 9-mer TFO. A detailed thermodynamic characterization of the unmodified TFO and its 5'-conjugate with a methyl-substituted ligand by ITC experiments yielded a DeltaDeltaG degrees of -1.8 kcal mol(-1) at pH 6.0 for the TFO-attached PIQ-triplex interaction and also revealed a favorable entropic contribution as the major determinant for the free energy of PIQ binding in the conjugate. The pH dependence of triplex thermal stability highlights the importance of ring protonation of the triplex-bound ligand for its effective interaction and triplex stabilization near physiological conditions.