Interactions of Binuclear Ruthenium(II) Complexes with Oligonucleotides in Hydrogel Matrix: Enantioselective Threading Intercalation into GC Context

A stretched poly­(vinyl alcohol) (PVA) film provides a unique matrix that enables also short DNA oligonucleotide duplex to be oriented and studied by linear dichroism (LD). This matrix further allows controlling DNA secondary structure by proper hydration (A or B form), and such humid films could potentially also mimic the molecular crowding in cellular contexts. However, early attempts to study intercalators and groove binders for probing DNA in PVA failed due to competitive matrix binding. Here we report the successful orientation in PVA of DNA oligonucleotide duplex hairpins with thread-intercalated binuclear complex [μ-(11,11′-bidppz)­(phen)₄Ru₂]⁴⁺, and how LD depends on oligonucleotide sequence and metal center chirality. Opposite enantiomers of the ruthenium complex, ΔΔ and ΛΛ, were investigated with respect to enantioselectivity toward GC stretches as long as 22 bp. LD, supported by emission kinetics, reveals that threading intercalation occurs only with ΔΔ whereas ΛΛ remains externally bound, probably in either or both of the grooves of the GC-DNA. Enantioselective binding properties of sterically rigid DNA probes such as the ruthenium complexes could find applications for targeting nucleic acids, e.g., to inhibit transcription in therapeutic context such as treatment of malaria or cancer

Minor-Groove Binding Drugs: Where Is the Second Hoechst 33258 Molecule?

Hoechst 33258 binds with high affinity into the minor groove of AT-rich sequences of double-helical DNA. Despite extensive studies of this and analogous DNA binding molecules, there still remains uncertainty concerning the interactions when multiple ligand molecules are accommodated within close distance. Albeit not of direct concern for most biomedical applications, which are at low drug concentrations, interaction studies for higher drug binding are important as they can give fundamental insight into binding mechanisms and specificity, including drug self-stacking interactions that can provide base-sequence specificity. Using circular dichroism (CD), isothermal titration calorimetry (ITC), and proton nuclear magnetic resonance (¹H NMR), we examine the binding of Hoechst 33258 to three oligonucleotide duplexes containing AT regions of different lengths: [d­(CGCGAATTCGCG)]₂ (A₂T₂), [d­(CGCAAATTTGCG)]₂ (A₃T₃), and [d­(CGAAAATTTTCG)]₂ (A₄T₄). We find similar binding geometries in the minor groove for all oligonucleotides when the ligand-to-duplex ratio is less than 1:1. At higher ratios, a second ligand can be accommodated in the minor groove of A₄T₄ but not A₂T₂ or A₃T₃. We conclude that the binding of the second Hoechst to A₄T₄ is not cooperative and that the molecules are sitting with a small separation apart, one after the other, and not in a sandwich structure as previously proposed

Enhanced Cellular Uptake of Antisecretory Peptide AF-16 through Proteoglycan Binding

Peptide AF-16, which includes the active site of Antisecretory Factor protein, has antisecretory and anti-inflammatory properties, making it a potent drug candidate for treatment of secretory and inflammatory diseases such as diarrhea, inflammatory bowel diseases, and intracranial hypertension. Despite remarkable physiological effects and great pharmaceutical need for drug discovery, very little is yet understood about AF-16 mechanism of action. In order to address interaction mechanisms, we investigated the binding of AF-16 to sulfated glycosaminoglycan, heparin, with focus on the effect of pH and ionic strength, and studied the influence of cell-surface proteoglycans on cellular uptake efficiency. Confocal laser scanning microscopy and flow cytometry experiments on wild type and proteoglycan-deficient Chinese hamster ovary cells reveal an endocytotic nature of AF-16 cellular uptake that is, however, less efficient for the cells lacking cell-surface proteoglycans. Isothermal titration calorimetry provides quantitative thermodynamic data and evidence for that the peptide affinity to heparin increases at lower pH and ionic strength. Experimental data, supported by theoretical modeling, of peptide–glycosaminoglycan interaction indicate that it has a large electrostatic contribution, which will be enhanced in diseases accompanied by decreased pH and ionic strength. These observations show that cell-surface proteoglycans are of general and crucial importance for the antisecretory and anti-inflammatory activities of AF-16

Structure of potassium salt of the acetic acid derivative of tC (KtC) upper left, tC nucleoside (tCnuc) upper right and G:tC base pair below

<p><b>Copyright information:</b></p><p>Taken from "Fluorescent properties of DNA base analogue tC upon incorporation into DNA — negligible influence of neighbouring bases on fluorescence quantum yield"</p><p>Nucleic Acids Research 2005;33(16):5019-5025.</p><p>Published online 7 Sep 2005</p><p>PMCID:PMC1201328.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p