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At least 217 records · Page 12Linked to original sources

PubMed2000

Studies on the fluorescence reaction between nucleic acid and the complex of cobalt(II) with 5-(3-fluoro-4-chlorophenylazo)-8-sulfonamidoquinoline and its applications.

Based on the enhancement of the fluorescence intensity of the complex of 5-(3-fluoro-4-chlorophenylazo)-8-sulfonamidoquinoline (FCPBSQ) with Co(2+) by nucleic acid in the presence of Tween 80 and in the weakly basic medium, a fluorescence method for the determination of nucleic acid was proposed. The calibration graphs for the determination of denatured calf thymus DNA (ct DNA), fish sperm DNA (fs DNA), and yeast RNA (yt RNA) were obtained in concentration ranges 0.050-4.0, 0.10-3.0, and 0.050-3.0 microg/ml with limits of detection of 0.010, 0.030, and 0.020 microg/ml, respectively. The method has been satisfactorily used for the determination of DNA in wheat cell extraction and ct DNA, fs DNA, and yt RNA in synthetic samples. Investigations on the binding mode by the Scatchard plots method suggested that both intercalation and electrostatic binding modes existed in this system.

Animals↗

PubMed1984

[Interaction of DNA and RNA ligases of phage T4 with Cibacron Blue F 3GA].

Using kinetic methods and differential spectrophotometry, the interaction between DNA and RNA ligases T4 and cibacron blue F3GA was studied. It was shown that the dye inhibits the first step of the enzymatic reaction, i. e. the formation of the AMP ligase complex. A 50% inhibition of the AMP-ligase complex by DNA ligase occurs at the dye concentration of 1 X 10(-5) M, that by RNA ligase--at 1 X 10(-4) M. Cibacron blue F3GA also inhibits the formation of end products of the reaction catalyzed by these enzymes. The dye is a noncompetitive inhibitor of RNA ligase with respect to [32P]oligoA20 and a competitive one with respect to ATP. Using differential spectrophotometry, it was found that the interaction occurs predominantly via electrostatic bonds between the SH-groups of the dye and the amino groups of lysine residues. DNA ligase possesses a higher affinity for the dye than RNA ligase.

Adenosine Triphosphate↗

PubMed2001

Determination of nucleic acids using calcein-neodymium complex as a fluorescence probe.

A novel fluorometric method has been developed for rapid determination of DNA and RNA with calcein-neodymium complex as a fluorescence probe. The method is based on the fluorescence enhancement of calcein-Nd(III) complex in the presence of DNA or RNA, with maximum excitation and emission wavelength at 489 nm and 514 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range 0.5 - 3.0 microg/ml for both DNA and yeast RNA, 0.4 - 2.0 microg/ml for fish sperm DNA (FS DNA) and 0 - 3.0 microg/ml for calf thymus DNA (CT DNA). The corresponding detection limits are 15.1 ng/ml for DNA, 21.2 ng/ml for yeast RNA, 10.5 ng/ml for FS DNA and 8.9 ng/ml for CT DNA. The interaction mechanism for the binding of calcein-Nd(III) complex to DNA is also studied. The results of absorption spectra, fluorescence polarization measurements and thermal denaturation experiments, suggested that the interaction between calcein-Nd(III) complex and DNA is an electrostatic interaction.

Animals↗

PubMed1998

Antibiotic inhibition of RNA catalysis: neomycin B binds to the catalytic core of the td group I intron displacing essential metal ions.

The aminoglycoside antibiotic neomycin B induces misreading of the genetic code during translation and inhibits several ribozymes. The self-splicing group I intron derived from the T4 phage thymidylate synthase (td) gene is one of these. Here we report how neomycin B binds to the intron RNA inhibiting splicing in vitro. Footprinting experiments identified two major regions of protection by neomycin B: one in the internal loop between the stems P4 and P5 and the other in the catalytic core close to the G-binding site. Mutational analyses defined the latter as the inhibitory site. Splicing inhibition is strongly dependent on pH and Mg2+ concentration, suggesting electrostatic interactions and competition with divalent metal ions. Fe2+-induced hydroxyl radical (Fe-OH.) cleavage of the RNA backbone was used to monitor neomycin-mediated changes in the proximity of the metal ions. Neomycin B protected several positions in the catalytic core from Fe-OH. cleavage, suggesting that metal ions are displaced in the presence of the antibiotic. Mutation of the bulged nucleotide in the P7 stem, a position which is strongly protected by neomycin B from Fe-OH. cleavage and which has been proposed to be involved in binding an essential metal ion, renders splicing resistant to neomycin. These results allowed the docking of neomycin to the core of the group I intron in the 3D model.

Anti-Bacterial Agents↗

PubMed1996

Strandedness discrimination in peptide-polynucleotide complexes.

Preferential binding to single- or double-stranded nucleic acids is important for the activity of many proteins that process RNA and DNA. We have investigated the mechanism of strandedness discrimination with peptides derived from the putative DNA-binding domain of the RecA protein, a bacterial recombinase that modulates its affinity for single-stranded DNA by means of ATP binding and hydrolysis. Contributions of electrostatic and non-electrostatic interactions to binding of these peptides with polynucleotides were evaluated by fluorescence spectroscopy as a function of salt concentration and peptide charge. Binding of these peptides to single- and double-stranded nucleic acids was dominated by non-electrostatic interactions. Small electrostatic contributions selectively enhanced peptide complexation with single-stranded nucleic acids. Similar results were observed in control experiments carried out with tripeptides containing charged and aromatic amino acid residues. It was possible to modify the strandedness preference of peptide-polynucleotide complexes by changing electrostatic contributions to the binding free energy. These observations suggest a mechanism whereby some proteins that interact with DNA or RNA might determine and regulate their relative affinity for single- and double-stranded nucleic acids.

Amino Acid Sequence↗

PubMedDate not supplied

DNA bending induced by carbocyclic sugar analogs constrained to the north conformation.

DNA bending caused by introduction of carbocyclic sugars constrained to the north conformation was studied, using explicit solvent molecular dynamic (MD) simulations. The native Drew-Dickerson (DD) dodecamer and its three modifications containing north carbocyclic sugars in the 7th (T7*), 8th (T8*) or both 7th and 8th (T7T8*) nucleotide positions were examined. Introduction of the carbocyclic sugar results in A-form conformations for the alpha, beta, chi, zeta, and sugar pucker backbone parameters in the modified nucleotides. Increased steric repulsion between the sugar and its parent base in the modified oligonucleotides impacts the roll and cup dinucleotide step parameters, increasing the bending of the oligomer axis. Increased buckling of the substituted nucleotides disrupts the usual stabilizing base stacking interactions. The level of overall bending depends on the number and position of carbocyclic sugars introduced in the DNA sequence. Single sugar substitutions are unable to induce substantial bending due to the neighboring unmodified nucleotides counterbalancing the distortion. Significant bending can, however, be induced by two consecutive north sugars (T7T8*), which is in agreement with experimental results. The modified oligomers populate a wide range of bend angles, indicating that they maintain flexibility in the bent state. The present results suggest that insertion of carbocyclic sugars into DNA or RNA duplexes can be used to engineer bending of the duplexes without impacting the electrostatic or chemical properties of the phosphodiester backbone, thereby serving as excellent tools for experimental elucidation of nucleic acid structure-function relationships.

Base Pairing↗

PubMed1998

Titration calculations of foot-and-mouth disease virus capsids and their stabilities as a function of pH.

Foot-and-mouth disease virus (FMDV), a non-enveloped picornavirus, is sensitive to acidic conditions. At pH values below 7 the icosahedral virus capsid, formed from 60 copies of a protomer containing four polypeptides (VP1 to 4), dissociates into 12 pentamers, releasing the viral RNA. Evidence suggests that this acid lability may assist FMDV cell entry via an endosomal pathway. Calculations of titration curves and pH-stability profiles are presented for three different strains of FMDV, O1BFS, A10(61) and A22 Iraq, and compared with experimental data for complete virions and empty capsids (which lack RNA). The finite difference Poisson-Boltzmann method was used for the calculation of electrostatic free energies with the solvent treated as a dielectric continuum. The inter-pentamer interface in the virus is formed by two protomers related by 2-fold icosahedral symmetry. As a simple model for inter-pentamer interactions, a dimer and two separate protomers were compared. The association free energy was computed by integrating the difference between the titration curves of the two species. The calculations reproduced the observed decrease in capsid stability at acidic pH but not the difference in pH sensitivities of the two type A viruses. It is shown that only residues within 15 A of the interface play a significant role in determining acid lability. For the experimentally studied pH range (5 to 7.6), histidine residues were found to dominate the pH-dependence of the stability. Two histidine residues in VP3, H142 and H145, are shown to have the greatest effect by virtue of their interactions with many polar residues across the inter-pentamer interface; the interaction of H142 with an alpha-helix in the opposite pentamer contributes only a small proportion of the destabilization energy.

Aphthovirus↗

PubMed2003

MolSurfer: A macromolecular interface navigator.

We describe the current status of the Java molecular graphics tool, MolSurfer. MolSurfer has been designed to assist the analysis of the structures and physico-chemical properties of macromolecular interfaces. MolSurfer provides a coupled display of two-dimensional (2D) maps of the interfaces generated with the ADS software and a three-dimensional (3D) view of the macromolecular structure in the Java PDB viewer, WebMol. The interfaces are analytically defined and properties such as electrostatic potential or hydrophobicity are projected on to them. MolSurfer has been applied previously to analyze a set of 39 protein-protein complexes, with structures available from the Protein Data Bank (PDB). A new application, described here, is the visualization of 75 interfaces in structures of protein-DNA and protein-RNA complexes. Another new feature is that the MolSurfer web server is now able to compute and map Poisson-Boltzmann electrostatic potentials of macromolecules onto interfaces. The MolSurfer web server is available at http://projects.villa-bosch.de/mcm/software/molsurfer.

Computer Graphics↗

PubMed2006

A novel layer-by-layer approach for the fabrication of conducting polymer/RNA multilayer films for controlled release.

Poly(anilineboronic acid) (PABA)/ribonucleic acid (RNA) multilayer films were prepared under neutral condition using a layer-by-layer deposition of PABA and RNA. RNA was used both as a polyelectrolyte for multilayer formation as well as dopant for PABA. Photoelastic modulated infrared reflection absorption spectroscopy measurements suggest that PABA interacts covalently with RNA through the formation of a boronate ester, a boron-nitrogen dative bond, as well as electrostatic interactions of anionic phosphates with cationic amines. The deposition procedure was monitored with UV-vis absorption spectroscopy, showing a linear dependence of absorbance with the number of PABA/RNA bilayers deposited. The multilayer films were further characterized using X-ray photoelectron spectroscopy and ellipsometry, which yielded a PABA/RNA bilayer thickness of approximately 10 nm. The PABA/RNA multilayer films are redox-active at neutral pH, consistent with the formation of a self-doped polymer. Electrochemical control of PABA under these conditions allows potential-induced controlled release of RNA from a multilayer at neutral pH, suggesting that this may serve as a novel method for controlled release of RNA under physiological conditions.

Aniline Compounds↗

PubMed2006

On structural transitions, thermodynamic equilibrium, and the phase diagram of DNA and RNA duplexes under torque and tension.

A precise understanding of the flexibility of double stranded nucleic acids and the nature of their deformed conformations induced by external forces is important for a wide range of biological processes including transcriptional regulation, supercoil and catenane removal, and site-specific recombination. We present, at atomic resolution, a simulation of the dynamics involved in the transitions from B-DNA and A-RNA to Pauling (P) forms and to denatured states driven by application of external torque and tension. We then calculate the free energy profile along a B- to P-transition coordinate and from it, compute a reversible pathway, i.e., an isotherm of tension and torque pairs required to maintain P-DNA in equilibrium. The reversible isotherm maps correctly onto a phase diagram derived from single molecule experiments, and yields values of elongation, twist, and twist-stretch coupling in agreement with measured values. We also show that configurational entropy compensates significantly for the large electrostatic energy increase due to closer-packed P backbones. A similar set of simulations applied to RNA are used to predict a novel structure, P-RNA, with its associated free energy, equilibrium tension, torque and structural parameters, and to assign the location, on the phase-diagram, of a putative force-torque-dependent RNA "triple point."

DNA↗

PubMed1996

Transcriptional activator of phage phi 29 late promoter: mapping of residues involved in interaction with RNA polymerase and in DNA bending.

Phage phi 29 regulatory protein p4 activates transcription from the late A3 promoter by stabilizing sigma A-RNA polymerase at the promoter as a closed complex. Activation requires interaction between both proteins. Protein p4 bends the DNA upon binding. We have performed a detailed mutagenesis study of the carboxyl end of the protein, which is involved in both transcription activation and DNA bending. The results indicate that Arg-120 is the most critical residue for activation, probably mediating the interaction with RNA polymerase. Several basic residues have been identified, including Arg-120, that contribute to maintenance of the DNA bending, probably via electrostatic interactions with the DNA backbone. The degree or stability of the induced bend apparently relies on the additive contribution of all basic residues of the carboxyl end of the protein. Therefore, the activation and DNA bending surfaces overlap, and Arg-120 should interact with both DNA and RNA polymerase. As we show that protein p4 is a dimer in solution, and is bound to DNA as a tetramer, the results suggest a model in which two of the p4 subunits interact with the DNA, bending it, while the other two subunits remain accessible to interact with RNA polymerase.

Bacillus Phages↗

PubMed1995

Functional characteristics of the maize RNA-binding protein MA16.

The maize RNA-binding protein MA16 is a non-ribosomal nucleolar protein widely distributed in different maize tissues. We have previously shown that the MA16 protein binds preferentially to guanosine- and uridine-rich sequences. As a step towards the identification of specific targets with which MA16 interacts within the cell, we investigated the RNA-binding affinities and several other aspects of the protein by using binding assays and immunochemistry. The MA16 protein showed a wide spectrum of RNA-binding activities with lower affinities to several RNAs that was salt and heparin-sensitive indicative of electrostatic interactions, and higher affinities to particular RNAs including rRNA and translatable mRNA sequences. Among the RNAs found associated with MA16 protein was that encoding MA16 itself. This observation raises the possibility that MA16 gene expression could be self-regulated. Immunoprecipitation studies showed that in vivo MA16 was phosphorylated and that MA16 interacts with RNAs through complex association with several proteins. These results suggest that both phosphorylation and interaction with other proteins may be involved in determining RNA-binding specificities of MA16 in the cell.

Heat-Shock Proteins↗

PubMed2003

Using pyrene-labeled HIV-1 TAR to measure RNA-small molecule binding.

To quantitatively understand the binding affinity and target selectivity of small-molecule RNA interactions, it is useful to have a rapid, highly reproducible binding assay that can be readily generalized to different RNA targets. To that end, an assay has been developed and validated for measuring the binding of low-molecular weight ligands to RNA by monitoring the fluorescence of a covalently incorporated fluorophore. As a test system, the fluorescence of a pyrene-derivatized HIV-1 TAR (transactivating response element) RNA was measured upon titration with aminoglycoside antibiotics. The binding isotherms thus obtained fit well with a model for a 1:1 interaction and yield an accurate measure of the equilibrium dissociation constant. Among a series of natural aminoglycosides, the binding affinity correlates with the number of amines, supporting an electrostatic compensation model for binding. Furthermore, the ionic strength dependence confirms that much of the binding energy is electrostatic. Finally, by measuring the binding affinity in the presence of nucleic acid competitors, we confirm that although aminoglycosides show high RNA to DNA selectivity, their selectivity among different RNA targets is sub- optimal. We conclude that this newly developed assay can be generalized to measure the binding affinities and selectivities of a variety of small molecules to a specific RNA target.

Aminoglycosides↗

PubMed1998

RNA as a drug target: chemical, modelling, and evolutionary tools.

Dramatic technical progress in RNA synthesis and structure determination has allowed several difficulties inherent to the preparation, handling and structural analysis of RNA to be overcome, and this has led to a wealth of information about RNA structure and its relationship with biological function. It is now fully recognized that RNA molecules intervene at all stages of cell life, not only because of key sequence motifs but also because of intricate three-dimensional folds. This realization has promoted RNA to a potential therapeutic target. As in protein motifs recognizing nucleic acids, groups of the molecule interacting with RNA contribute to specific binding through defined hydrogen bonds and van der Waals docking, while other parts contribute to the driving force of binding via less specific electrostatic interactions accompanied by water and ion displacement.

Aminoglycosides↗

PubMed2004

Principles of RNA compaction: insights from the equilibrium folding pathway of the P4-P6 RNA domain in monovalent cations.

Counterions are required for RNA folding, and divalent metal ions such as Mg(2+) are often critical. To dissect the role of counterions, we have compared global and local folding of wild-type and mutant variants of P4-P6 RNA derived from the Tetrahymena group I ribozyme in monovalent and in divalent metal ions. A remarkably simple picture of the folding thermodynamics emerges. The equilibrium folding pathway in monovalent ions displays two phases. In the first phase, RNA molecules that are initially in an extended conformation enforced by charge-charge repulsion are relaxed by electrostatic screening to a state with increased flexibility but without formation of long-range tertiary contacts. At higher concentrations of monovalent ions, a state that is nearly identical to the native folded state in the presence of Mg(2+) is formed, with tertiary contacts that involve base and backbone interactions but without the subset of interactions that involve specific divalent metal ion-binding sites. The folding model derived from these and previous results provides a robust framework for understanding the equilibrium and kinetic folding of RNA.

Animals↗

PubMed1997

The refined crystal structure of the 3C gene product from hepatitis A virus: specific proteinase activity and RNA recognition.

The virally encoded 3C proteinases of picornaviruses process the polyprotein produced by the translation of polycistronic viral mRNA. The X-ray crystallographic structure of a catalytically active mutant of the hepatitis A virus (HAV) 3C proteinase (C24S) has been determined. Crystals of this mutant of HAV 3C are triclinic with unit cell dimensions a = 53.6 A, b = 53.5 A, c = 53.2 A, alpha = 99.1 degrees, beta = 129.0 degrees, and gamma = 103.3 degrees. There are two molecules of HAV 3C in the unit cell of this crystal form. The structure has been refined to an R factor of 0.211 (Rfree = 0.265) at 2.0-A resolution. Both molecules fold into the characteristic two-domain structure of the chymotrypsin-like serine proteinases. The active-site and substrate-binding regions are located in a surface groove between the two beta-barrel domains. The catalytic Cys 172 S(gamma) and His 44 N(epsilon2) are separated by 3.9 A; the oxyanion hole adopts the same conformation as that seen in the serine proteinases. The side chain of Asp 84, the residue expected to form the third member of the catalytic triad, is pointed away from the side chain of His 44 and is locked in an ion pair interaction with the epsilon-amino group of Lys 202. A water molecule is hydrogen bonded to His 44 N(delta1). The side-chain phenolic hydroxyl group of Tyr 143 is close to this water and to His 44 N(delta1) and may be negatively charged. The glutamine specificity for P1 residues of substrate cleavage sites is attributed to the presence of a highly conserved His 191 in the S1 pocket. A very unusual environment of two water molecules and a buried glutamate contribute to the imidazole tautomer believed to be important in the P1 specificity. HAV 3C proteinase has the conserved RNA recognition sequence KFRDI located in the interdomain connection loop on the side of the molecule diametrically opposite the proteolytic site. This segment of polypeptide is located between the N- and C-terminal helices, and its conformation results in the formation of a well-defined surface with a strongly charged electrostatic potential. Presumably, this surface of HAV 3C participates in the recognition of the 5' and 3' nontranslated regions of the RNA genome during viral replication.

3C Viral Proteases↗

PubMed1997

[New strategy for RNA vectorization in mammalian cells. Use of a peptide vector].

A major barrier for gene delivery is the low permeability of nucleic acids to cellular membranes. The development of antisenses and gene therapy has focused mainly on improving methods of oligonucleotide or gene delivery to the cell. In this report we described a new strategy for RNA cell delivery, based on a short single peptide. This peptide vector is derived from both the fusion domain of the gp41 protein of HIV and the nuclear localization sequence of the SV40 large T antigen. This peptide vector localizes rapidly to the cytoplasm then to the nucleus of human fibroblasts (HS-68) within a few minutes and exhibits a high affinity for a single-stranded mRNA encoding the p66 subunit of the HIV-1 reverse transcriptase (in a 100 nM range). The peptide/RNA complex formation involves mainly electrostatic interactions between the basic residues of the peptide and the charges on the phosphate group of the RNA. In the presence of the peptide-vector fluorescently-labelled mRNA is delivered into the cytoplasm of mammalian cells (HS68 human fibroblasts) in less than 1 h with a relatively high efficiency (80%). This new concept based on a peptide-derived vector offers several advantages compared to other compounds commonly used in gene delivery. This vector is highly soluble and exhibits no cytotoxicity at the concentrations used for optimal gene delivery. This result clearly supports the fact that this peptide vector is a powerful tool and that it can be used widely, as much for laboratory research as for new applications and development in gene and/or antisense therapy.

Cells, Cultured↗

PubMed1995

Structures of the native and swollen forms of cowpea chlorotic mottle virus determined by X-ray crystallography and cryo-electron microscopy.

BACKGROUND: RNA-protein interactions stabilize many viruses and also the nucleoprotein cores of enveloped animal viruses (e.g. retroviruses). The nucleoprotein particles are frequently pleomorphic and generally unstable due to the lack of strong protein-protein interactions in their capsids. Principles governing their structures are unknown because crystals of such nucleoprotein particles that diffract to high resolution have not previously been produced. Cowpea chlorotic mottle virions (CCMV) are typical of particles stabilized by RNA-protein interactions and it has been found that crystals that diffract beyond 4.5 A resolution are difficult to grow. However, we report here the purification of CCMV with an exceptionally mild procedure and the growth of crystals that diffract X-rays to 3.2 A resolution. RESULTS: The 3.2 A X-ray structure of native CCMV, an icosahedral (T = 3) RNA plant virus, shows novel quaternary structure interactions based on interwoven carboxyterminal polypeptides that extend from canonical capsid beta-barrel subunits. Additional particle stability is provided by intercapsomere contacts between metal ion mediated carboxyl cages and by protein interactions with regions of ordered RNA. The structure of a metal-free, swollen form of the virus was determined by cryo-electron microscopy and image reconstruction. Modeling of this structure with the X-ray coordinates of the native subunits shows that the 29 A radial expansion is due to electrostatic repulsion at the carboxyl cages and is stopped short of complete disassembly by preservation of interwoven carboxyl termini and protein-RNA contacts. CONCLUSIONS: The CCMV capsid displays quaternary structural interactions that are unique compared with previously determined RNA virus structures. The loosely coupled hexamer and pentamer morphological units readily explain their versatile reassembly properties and the pH and metal ion dependent polymorphism observed in the virions. Association of capsomeres through inter-penetrating carboxy-terminal portions of the subunit polypeptides has been previously described only for the DNA tumor viruses, SV40 and polyoma.

Amino Acid Sequence↗