Circular dichroism and conformational polymorphism of DNA

Here we review studies that provided important information about conformational properties of DNA using circular dichroic (CD) spectroscopy. The conformational properties include the B-family of structures, A-form, Z-form, guanine quadruplexes, cytosine quadruplexes, triplexes and other less characterized structures. CD spectroscopy is extremely sensitive and relatively inexpensive. This fast and simple method can be used at low- as well as high-DNA concentrations and with short- as well as long-DNA molecules. The samples can easily be titrated with various agents to cause conformational isomerizations of DNA. The course of detected CD spectral changes makes possible to distinguish between gradual changes within a single DNA conformation and cooperative isomerizations between discrete structural states. It enables measuring kinetics of the appearance of particular conformers and determination of their thermodynamic parameters. In careful hands, CD spectroscopy is a valuable tool for mapping conformational properties of particular DNA molecules. Due to its numerous advantages, CD spectroscopy significantly participated in all basic conformational findings on DNA

Arrangements of human telomere DNA quadruplex in physiologically relevant K+ solutions

The arrangement of the human telomeric quadruplex in physiologically relevant conditions has not yet been unambiguously determined. Our spectroscopic results suggest that the core quadruplex sequence G3(TTAG3)3 forms an antiparallel quadruplex of the same basket type in solution containing either K+ or Na+ ions. Analogous sequences extended by flanking nucleotides form a mixture of the antiparallel and hybrid (3 + 1) quadruplexes in K+-containing solutions. We, however, show that long telomeric DNA behaves in the same way as the basic G3(TTAG3)3 motif. Both G3(TTAG3)3 and long telomeric DNA are also able to adopt the (3 + 1) quadruplex structure: Molecular crowding conditions, simulated here by ethanol, induced a slow transition of the K+-stabilized quadruplex into the hybrid quadruplex structure and then into a parallel quadruplex arrangement at increased temperatures. Most importantly, we demonstrate that the same transitions can be induced even in aqueous, K+-containing solution by increasing the DNA concentration. This is why distinct quadruplex structures were detected for AG3(TTAG3)3 by X-ray, nuclear magnetic resonance and circular dichrosim spectroscopy: Depending on DNA concentration, the human telomeric DNA can adopt the antiparallel quadruplex, the (3 + 1) structure, or the parallel quadruplex in physiologically relevant concentrations of K+ ions

Structural characterization of cationic lipid–tRNA complexes

Despite considerable interest and investigations on cationic lipid–DNA complexes, reports on lipid–RNA interaction are very limited. In contrast to lipid–DNA complexes where lipid binding induces partial B to A and B to C conformational changes, lipid–tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid–tRNA interactions with G–C and A–U base pairs as well as the backbone phosphate group with overall binding constants of KChol = 5.94 (± 0.8) × 104 M–1, KDDAB = 8.33 (± 0.90) × 105 M–1, KDOTAP = 1.05 (± 0.30) × 105 M–1 and KDOPE = 2.75 (± 0.50) × 104 M–1. The order of stability of lipid–tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations

Structural characterization of cationic lipid–tRNA complexes

Despite considerable interest and investigations on cationic lipid–DNA complexes, reports on lipid–RNA interaction are very limited. In contrast to lipid–DNA complexes where lipid binding induces partial B to A and B to C conformational changes, lipid–tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid–tRNA interactions with G–C and A–U base pairs as well as the backbone phosphate group with overall binding constants of KChol = 5.94 (± 0.8) × 104 M–1, KDDAB = 8.33 (± 0.90) × 105 M–1, KDOTAP = 1.05 (± 0.30) × 105 M–1 and KDOPE = 2.75 (± 0.50) × 104 M–1. The order of stability of lipid–tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations

The effect of the TRF2 N-terminal and TRFH regions on telomeric G-quadruplex structures

The sequence of human telomeric DNA consists of tandem repeats of 5′-d(TTAGGG)-3′. This guanine-rich DNA can form G-quadruplex secondary structures which may affect telomere maintenance. A current model for telomere protection by the telomere-binding protein, TRF2, involves the formation of a t-loop which is stabilized by a strand invasion-like reaction. This type of reaction may be affected by G-quadruplex structures. We analyzed the influence of the arginine-rich, TRF2 N-terminus (TRF2B), as well as this region plus the TRFH domain of TRF2 (TRF2BH), on the structure of G-quadruplexes. Circular dichroism results suggest that oligonucleotides with 4, 7 and 8 5′-d(TTAGGG)-3′ repeats form hybrid structures, a mix of parallel/antiparallel strand orientation, in K+. TRF2B stimulated the formation of parallel-stranded structures and, in some cases, intermolecular structures. TRF2BH also stimulated intermolecular but not parallel-stranded structures. Only full-length TRF2 and TRF2BH stimulated uptake of a telomeric single-stranded oligonucleotide into a plasmid containing telomeric DNA in the presence of K+. The results in this study suggest that G-quadruplex formation inhibits oligonucleotide uptake into the plasmid, but the inhibition can be overcome by TRF2. This study is the first analysis of the effects of TRF2 domains on G-quadruplex structures and has implications for the role of G-quadruplexes and TRF2 in the formation of t-loops