Aminoglycoside complexation with a DNA.RNA hybrid duplex: the thermodynamics of recognition and inhibition of RNA processing enzymes

Spectroscopic and calorimetric techniques were employed to characterize and contrast the binding of the aminoglycoside paromomycin to three octamer nucleic acid duplexes of identical sequence but different strand composition (a DNA.RNA hybrid duplex and the corresponding DNA.DNA and RNA.RNA duplexes). In addition, the impact of paromomycin binding on both RNase H- and RNase A-mediated cleavage of the RNA strand in the DNA.RNA duplex was also determined. Our results reveal the following significant features: (i) Paromomycin binding enhances the thermal stabilities of the RNA.RNA and DNA.RNA duplexes to similar extents, with this thermal enhancement being substantially greater in magnitude than that of the DNA.DNA duplex. (ii) Paromomycin binding to the DNA.RNA hybrid duplex induces CD changes consistent with a shift from an A-like to a more canonical A-conformation. (iii) Paromomycin binding to all three octamer duplexes is linked to the uptake of a similar number of protons, with the magnitude of this number being dependent on pH. (iv) The affinity of paromomycin for the three host duplexes follows the hierarchy, RNA.RNA > DNA.RNA > DNA.DNA. (v) The observed affinity of paromomycin for the RNA.RNA and DNA.RNA duplexes decreases with increasing pH. (vi) The binding of paromomycin to the DNA.RNA hybrid duplex inhibits both RNase H- and RNase A-mediated cleavage of the RNA strand. We discuss the implications of our combined results with regard to the specific targeting of DNA.RNA hybrid duplex domains and potential antiretroviral applications.     

Size, shape, and flexibility of RNA structures

Determination of sizes and flexibilities of RNA molecules is important in understanding the nature of packing in folded structures and in elucidating interactions between RNA and DNA or proteins. Using the coordinates of the structures of RNA in the Protein Data Bank we find that the size of the folded RNA structures, measured using the radius of gyration R(G), follows the Flory scaling law, namely, R(G)=5.5N(1/3) A, where N is the number of nucleotides. The shape of RNA molecules is characterized by the asphericity Delta and the shape S parameters that are computed using the eigenvalues of the moment of inertia tensor. From the distribution of Delta, we find that a large fraction of folded RNA structures are aspherical and the distribution of S values shows that RNA molecules are prolate (S>0). The flexibility of folded structures is characterized by the persistence length l(p). By fitting the distance distribution function P(r), that is computed using the coordinates of the folded RNA, to the wormlike chain model we extracted the persistence length l(p). We find that l(p) approximately 1.5N(0.33) A which might reflect the large separation between the free energies that stabilize secondary and tertiary structures. The dependence of l(p) on N implies that the average length of helices should increase as the size of RNA grows. We also analyze packing in the structures of ribosomes (30S, 50S, and 70S) in terms of R(G), Delta, S, and l(p). The 70S and the 50S subunits are more spherical compared to most RNA molecules. The globularity in 50S is due to the presence of an unusually large number (compared to 30S subunit) of small helices that are stitched together by bulges and loops. Comparison of the shapes of the intact 70S ribosome and the constituent particles suggests that folding of the individual molecules might occur prior to assembly.     

Structure and properties of metal-exchanged zeolites studied using gradient-corrected and hybrid functionals. I. Structure and energetics

The structural and energetic properties of purely siliceous, proton-, and Cu- and Co-exchanged chabazite have been studied using periodic density-functional (DFT) calculations with both conventional gradient-corrected exchange-correlation functionals and hybrid functionals mixing exact (i.e., Hartree-Fock) and DFT exchange. Spin-polarized and fixed-moment calculations have been performed to determine the equilibrium and excited spin-configurations of the metal-exchanged chabazites. For the purely siliceous chabazite, hybrid functionals predict a slightly more accurate cell volume and lattice geometry. For isolated Al/Si substitution sites, gradient-corrected functionals predict that the lattice distortion induced by the substitution preserves the local tetrahedral symmetry, whereas hybrid functionals lead to a distorted Al coordination with two short and two long Al-O bonds. Hybrid functionals yield a stronger cation-framework binding that conventional functionals in metal-exchanged zeolites, they favor shorter cation-oxygen bonds and eventually also a higher coordination of the cation. Both types of functionals predict the same spin in the ground-state. The structural optimization of the excited spin-states shows that the formation of a high-spin configuration leads to a strong lattice relaxation and a weaker cation-framework bonding. For both Cu- and Co-exchanged chabazite, the prediction of a preferred location of the cation in a six-membered ring of the zeolite agrees with experiment, but the energy differences between possible cation locations and the lattice distortion induced by the Al/Si substitution and the bonding of the cation depends quite significantly on the choice of the functional. All functionals predict similar energy differences for excited spin states. Spin-excitations are shown to be accompanied by significant changes in the cation coordination, which are more pronounced with hybrid functionals. The consequences of electronic spectra and chemical reactivity are analyzed in the following papers.     

Circular DNA and DNA/RNA hybrid molecules as scaffolds for ricin inhibitor design

Ricin Toxin A-chain (RTA) catalyzes the hydrolytic depurination of A4324, the first adenosine of the GAGA tetra-loop portion of 28S eukaryotic ribosomal RNA. Truncated stem-loop versions of the 28S rRNA are RTA substrates. Here, we investigate circular DNA and DNA/RNA hybrid GAGA sequence oligonucleotides as minimal substrates and inhibitor scaffolds for RTA catalysis. Closing the 5'- and 3'-ends of a d(GAGA) tetraloop creates a substrate with 92-fold more activity with RTA (kcat/Km) than that for the d(GAGA) linear form. Circular substrates have catalytic rates (kcat) comparable to and exceeding those of RNA and DNA stem-loop substrates, respectively. RTA inhibition into the nanomolar range has been achieved by introducing an N-benzyl-hydroxypyrrolidine (N-Bn) transition state analogue at the RTA depurination site in a circular GAGA motif. The RNA/DNA hybrid oligonucleotide cyclic GdAGA provides a new scaffold for RTA inhibitor design, and cyclic G(N-Bn)GA is the smallest tight-binding RTA inhibitor (Ki = 70 nM). The design of such molecules that lack the base-paired stem-loop architecture opens new chemical synthetic approaches to RTA inhibition.     

Conformational flexibility of DNA

Pulsed Electron-Electron Double Resonance (PELDOR) on double-stranded DNA (ds-DNA) was used to investigate the conformational flexibility of helical DNA. Stretching, twisting, and bending flexibility of ds-DNA was determined by incorporation of two rigid nitroxide spin labels into a series of 20 base pair (bp) DNA duplexes. Orientation-selective PELDOR experiments performed at both X-band (9 GHz/0.3 T) and G-band (180 GHz/6.4 T) with spin label distances in the range of 2-4 nm allowed us to differentiate between different simple models of DNA dynamics existing in the literature. All of our experimental results are in full agreement with a dynamic model for ds-DNA molecules, where stretching of the molecule leads to a slightly reduced radius of the helix induced by a cooperative twist-stretch coupling.     

Tuning urea-phenanthridinium conjugates for DNA/RNA and base pair recognition

A series of novel urea-phenanthridine conjugates was prepared. The variation of linker length connecting two urea-phenanthridinium conjugates regulated their binding mode toward double stranded polynucleotides, consequently controlling selectivity of compounds toward ds-RNA over ds-DNA stabilization as well as selective fluorescence response toward addition of G-C base pair and A-U(T) base pair containing polynucleotides.     

Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA

Locked nucleic acid is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2'-oxygen and the 4'-carbon. This conformation restriction increases binding affinity for complementarity sequences and provides an exciting new chemical approach for the control of gene expression and optimization of microarrays.     

Electrochemical properties of niosomes modified Au electrode and DNA recognition

Non-ionic surfactant vesicles (NSVs), also referred to as niosomes, have been studied as an alternative to conventional liposomes. In this paper, electrochemical inspection of the interaction between Herring sperm DNA and niosomes has been investigated after a simple and novel method for the formation of niosomes on Au electrode. Each step of electrode modification has been confirmed with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The assembly of octadecanethiol (ODT) layer on the electrode surface generates a packed film that introduces a barrier to the interfacial electron transfer (R_et), and the subsequent immobilization of niosomes onto the self-assembled monolayer (SAM) layer results in a further increase of R_et, due to the formed bilayer almost blocked the redox probe to the electrode surface. When Herring sperm DNA was added, the R_et value decreased, indicating that the barrier of the redox probe to the surface was disrupted. The addition of DNA caused the formation of some transmembrane channels for the redox probe across the niosomes. A good linear relationship between R_et value and DNA concentration was found over the 0–0.05 mg mL⁻¹ concentration range.     

Electrochemical properties of niosomes modified Au electrode and DNA recognition

Non-ionic surfactant vesicles (NSVs), also referred to as niosomes, have been studied as an alternative to conventional liposomes. In this paper, electrochemical inspection of the interaction between Herring sperm DNA and niosomes has been investigated after a simple and novel method for the formation of niosomes on Au electrode. Each step of electrode modification has been confirmed with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The assembly of octadecanethiol (ODT) layer on the electrode surface generates a packed film that introduces a barrier to the interfacial electron transfer (R(et)), and the subsequent immobilization of niosomes onto the self-assembled monolayer (SAM) layer results in a further increase of R(et), due to the formed bilayer almost blocked the redox probe to the electrode surface. When Herring sperm DNA was added, the R(et) value decreased, indicating that the barrier of the redox probe to the surface was disrupted. The addition of DNA caused the formation of some transmembrane channels for the redox probe across the niosomes. A good linear relationship between R(et) value and DNA concentration was found over the 0-0.05 mg mL(-1) concentration range.     

Structure and conformational flexibility of hexamethyldiperoxacyclononane

3,3,6,6,9,9-Hexamethyl-1,2,4,5-tetraoxacyclononane, one of the most thermally stable organic peroxides, has been studied by X-ray structural analysis. Possible conformations of this compound have been calculated by the molecular mechanics method.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 110–112, January, 1995.     

Electron affinities of the DNA and RNA bases.

Adiabatic electron affinities (AEAs) for the DNA and RNA bases are predicted by using a range of density functionals with a double-zeta plus polarization plus diffuse (DZP++) basis set in an effort to bracket the true EAs. Although the AEAs exhibit moderate fluctuations with respect to the choice of functional, systematic trends show that the covalent uracil (U) and thymine (T) anions are bound by 0.05-0.25 eV while the adenine (A) anion is clearly unbound. The computed AEAs for cytosine (C) and guanine (G) oscillate between small positive and negative values for the three most reliable functional combinations (BP86, B3LYP, and BLYP), and it remains unclear if either covalent anion is bound. AEAs with B3LYP/TZ2P++ single points are 0.19 (U), 0.16 (T), 0.07 (G), -0.02 (C), and -0.17 eV (A). Favorable comparisons are made to experimental estimates extrapolated from photoelectron spectra data for the complexes of the nucleobases with water. However, experimental values scaled from liquid-phase reduction potentials are shown to overestimate the AEAs by as much as 1.5 eV. Because the uracil and thymine covalent EAs are in energy ranges near those of their dipole-bound counterparts, preparation and precise experimental measurement of the thermodynamically stable covalent anions may prove challenging.     

Single-molecule studies on DNA and RNA.

DNA and RNA are the most individual molecules known. Therefore, single-molecule experiments with these nucleic acids are particularly useful. This review reports on recent experiments with single DNA and RNA molecules. First, techniques for their preparation and handling are summarised including the use of AFM nanotips and optical or magnetic tweezers. As important detection techniques, conventional and near-field microscopy as well as fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) are touched on briefly. The use of single-molecule techniques currently includes force measurements in stretched nucleic acids and in their complexes with binding partners, particularly proteins, and the analysis of DNA by restriction mapping, fragment sizing and single-molecule hybridisation. Also, the reactions of RNA polymerases and enzymes involved in DNA replication and repair are dealt with in some detail, followed by a discussion of the transport of individual nucleic acid molecules during the readout and use of genetic information and during the infection of cells by viruses. The final sections show how the enormous addressability in nucleic acid molecules can be exploited to construct a single-molecule field-effect transistor and a walking single-molecule robot, and how individual DNA molecules can be used to assemble a single-molecule DNA computer.     

Balancing flexibility and stress in DNA nanostructures

A subtle balance of flexibility and stress is found to be critical for a DNA nanostructure to be a good self-assembly block.     

Duplex stability of DNA.DNA and DNA.RNA duplexes containing 3'-S-phosphorothiolate linkages

3'-S-Phosphorothiolate (3'-SP) linkages have been incorporated into the DNA strand of both a DNA.RNA duplex and a DNA.DNA duplex. Thermal melting (T(m)) studies established that this modification significantly stabilises the DNA.RNA duplex with an average increase in T(m) of about 1.4 degrees C per modification. For two or three modifications, the increase in T(m) was larger for an alternating, as compared to the contiguous, arrangement. For more than three modifications their arrangement had no effect on T(m). In contrast to the DNA.RNA duplex, the 3'-S-phosphorothiolate linkage destabilised the DNA.DNA duplex, irrespective of the arrangement of the 3'-SP linkages. The effect of ionic strength on duplex stability was similar for both the phosphorothiolate-substituted and the unmodified RNA.DNA duplexes. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and comparisons are also drawn between the current study and those previously conducted with other modifications that have a similar conformational effect.