A comparison between the hot and cold water soluble fractions of two locust bean gum samples

Locust bean gum extracted from two carob flours from eastern and western Mediterranean sources were fractionated on the basis of their solubility in water. Weight-average molecular weights determined by sedimentation equilibrium were about 300 000 for both the hot water and cold water soluble fractions, whereas a commercial sample of guar gum had a molecular weight of 700 000. Their values were lower than would be predicted from Mark-Houwink relationships where molecular weights were originally determined by light scattering.

The hot water soluble fraction from the eastern Mediterranean flour showed unexpected rheological behaviour. It had an extremely high Huggins' constant and a different relationship between the coil overlap parameter and the zero shear rate viscosity compared with previously reported results for galactomannans. Both effects may be explained by the anomalously low intrinsic viscosity of this fraction when determined by a Huggins' extrapolation. The use of the Kraemer extrapolation gave significantly higher intrinsic viscosities for this particular sample. Gels formed from the two hot water soluble fractions with κ-carrageenan had similar rheological properties.     

Interactions of retroviral structural proteins with single-stranded nucleic acids

We have studied the interactions of single-stranded polyribonucleotides with murine leukemia virus structural proteins p10, p10' (a p10 variant), and Pr65gag, as well as Rous sarcoma virus (RSV) pp12 (a p10 analog). Two quantitative assays have been used to monitor protein-RNA association: the fluorescence enhancement of polyethenoadenylic acid) poly(epsilon A) upon binding protein, and tryptophan fluorescence quenching upon binding to poly(U). With each assay p10 was shown to bind stoichiometrically to single-stranded RNA, covering a length of nucleic acid chain (occluded site size, n) of about 6 residues. RSV pp12 was also shown to bind to poly(epsilon A), with n = 5 +/- 1. Addition of NaCl to fully titrated MuLV p10-nucleic acid mixtures effected nearly complete restoration of poly(epsilon A) or MuLV p10 fluorescence. Under conditions of 0.06 M NaCl, p10 bound noncooperatively to poly(epsilon A) with an intrinsic association constant, K = 2.3 X 10(6) M-1. K and n determined in this study were shown to relate to Kapp determined by other methods, by the approximation Kapp approximately NK, where N is the number of binding sites along the polynucleotide chain ((nucleotides/chain)/n). Chemical modifications of the p10 cysteine residues did not alter the affinity for poly(epsilon A). The affinity of Pr65gag for poly(epsilon A) appears to be higher than that of p10.     

Interaction of nucleolar phosphoprotein B23 with nucleic acids

The interaction of eukaryotic nucleolar phosphoprotein B23 with nucleic acids was examined by gel retardation and filter binding assays, by fluorescence techniques, and by circular dichroism. All studies utilized protein prepared under native conditions by a newly developed purification procedure. Electrophoretic gel mobility shift assays with phage M13 DNA suggested that protein B23 is a single-stranded nucleic acid binding protein. This was confirmed in competition binding assays with native or heat-denatured linearized plasmid pUC18 DNA where the protein showed a marked preference for the denatured form. In other competition assays, there was no apparent preference for single-stranded synthetic ribo- versus deoxyribonucleotides. Equilibrium binding with poly(riboethenoadenylic acid) indicated cooperative ligand binding with a protein binding site size of 11 nucleotides and an apparent binding constant (K omega) of 5 x 10(7) M-1 which includes an intrinsic binding constant (K) of 6.3 x 10(4) M-1 and a cooperativity factor (omega) of 800. In circular dichroism (CD) studies, protein B23, when combined with the single-stranded synthetic nucleic acids poly(rA) and poly(rC), effected a decrease in ellipticity and a shift of the positive peak at 260-270 nm toward higher wavelengths, indicating helix destabilizing activity. No CD changes were seen with double-stranded poly(dA.dT). The change in ellipticity of poly(rA) was sigmoidal upon addition of protein, confirming the cooperative behavior seen with fluorescence methods. These studies indicate that protein B23 binds cooperatively with high affinity for single-stranded nucleic acids and exhibits RNA helix destabilizing activity. These features may be related to its role in ribosome assembly.     

Estimating the Contributions of Selection and Self-Organization in RNA Secondary Structure

In addition to characteristic structural properties imposed by evolutionary modification, evolved, single-stranded RNAs also display characteristic structural properties imposed by intrinsic physical constraints on RNA polymer folding. The balance of intrinsic and functionally selected characters in the folded conformation of evolved secondary structures was determined by comparing the predicted secondary structures of evolved and unevolved (random) RNA sequences. Though evolved conformations are significantly more ordered than conformations of random-sequence RNA, this analysis demonstrates that the majority of conformational order within evolved structures results not from evolutionary optimization but from constraints imposed by rules intrinsic to RNA polymer folding. Received: 25 November 1998 / Accepted: 12 February 1999     

Crystal structure of an RNA aptamer bound to thrombin

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 A resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.     

Depolymerization of N-succinyl-chitosan by hydrochloric acid

N-Succinyl-chitosan (1) was depolymerized with 7.5 M aqueous HCl at room temperature or 3.3 M aqueous HCl at 40 degrees C and the molecular weights (MW) of the products were determined by size-exclusion chromatography-multi angle light scattering (SEC-MALS) and their viscometric features were investigated. The intrinsic viscosity ([eta]) obtained at the concentration of 0.1-0.3% (w/v) in saline showed a linear relationship between log[eta] and log MW, which provided the coefficients in the Mark-Houwink equation.     

Nucleic acid binding properties of Escherichia coli ribosomal protein S1. I. Structure and interactions of binding site I.

Escherichia coli ribosomal protein S1 plays a central role in initiation of protein synthesis, perhaps via participation in the binding of messenger RNA to the ribosome. S1 protein has two nucleic acid binding sites with very different properties: site I binds either single-stranded DNA or RNA, while site II binds single-stranded RNA only (Draper et al., 1977). The nucleic acid binding properties of these sites have been explored using the quenching of intrinsic protein fluorescence which results from binding of oligo- and polynucleotides, and are reported in this and the accompanying paper (Draper &; von Hippel, 1978).Site I has been studied primarily using DNA oligomers and polymers, and has been found to have the following properties. (1) The intrinsic binding constant (K) of site I for poly(dA) and poly(dC) is ~3 × 10⁶m^?1 at 0.12 m-Na⁺, and the site size (n, the number of nucleotide residues covered per S1 bound) is 5.1 ± 1.0 residues. (2) Binding of site I to polynucleotides is non-co-operative. (3) The K value for binding of S1 to single-stranded polynucleotides is ~10³ larger than K for binding to double-stranded polynucleotides, meaning that S1 (via site I) is a potential “melting” or “double-helix destabilizing” protein. (4) The dependence of log K on log [Na⁺] is linear, and analysis of the data according to Record et al. (1976) shows that two basic residues in site I form charge-charge interactions with two DNA phosphates. In addition, a major part of the binding free energy of site I with the nucleic acid chain appears to involve non-electrostatic interactions. (5) Oligonucleotides bound in site II somewhat weaken the binding affinity of site I. (6) Binding affin is virtually independent of base and sugar composition of the nucleic acid ligand; in fact, the total absence of the base appears to have little effect on the binding, since the association constant for 2′-deoxyribose 5′-phosphate is approximately the same as that for dAMP or dCMP. (7) Two molecules of d(ApA) can bind to site I, suggesting the presence of two “subsites” within site I. (8) Iodide quenching experiments with S1-oligonucleotide complexes show differential exposure of tryptophans in and near the subsites of site I, depending upon whether neither, one, or both subsites are complexed with an oligonucleotide.     

Replication of Sindbis virus. I. Relative size and genetic content of 26 s and 49 s RNA

The genome of Sindbis virus, 49 s RNA, is a single, intact polynucleotide chain having a molecular weight of 4.3±0.3 × 10⁶ daltons. This has been determined using a variety of methods including polyacrylamide gel electrophoresis, sedimentation after reaction with formaldehyde, determination of the molecular weights of the double-stranded forms of Sindbis-specific RNA and hybridization competition. The second major species of single-stranded RNA made after infection with Sindbis, 26 s RNA, has been found to have a molecular weight of 1.6 × 10⁶ daltons as determined by sedimentation in dimethylsulfoxide. Hybridizationcompetition experiments carried out between these two species of RNA, using double-stranded forms of Sindbis RNA isolated from infected cells, showed that 26 s RNA contains only one-third of the base sequences in 49 s RNA and thus represents a unique fraction of the viral genome.     

Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts.

We describe four monoclonal antibodies (MAB) which specifically recognize double-stranded RNA (dsRNA) together with their use in new methods for detecting and characterizing dsRNA in unfractionated nucleic acid extracts. The specificity of the antibodies was analyzed using a panel of 27 different synthetic and naturally occurring nucleic acids. All four antibodies reacted in a highly specific manner with long dsRNA helices, irrespective of their sequence; no binding to single-stranded RNA homopolymers or to DNA or RNA-DNA hybrids was observed. The apparent affinity of the antibodies to short (less than or equal to 11 bp) RNA helices was very low in all test systems used: only background levels of binding were obtained on single-stranded RNA species which contain double-helical secondary structures (e.g. rRNA, tRNA, viroid RNA). A sandwich ELISA and a dsRNA-immunoblotting procedure have been established which allow detection and characterization of dsRNA by MAB even in the presence of a large excess of other nucleic acids. In combination with temperature-gradient gelelectrophoresis (TGGE) not only the molecular weights but also the highly characteristic Tm-values of conformational transitions of individual dsRNA species could be determined by immunoblotting. An example of the general use of these methods for the detection of plant virus infections is demonstrated with groundnut rosette virus (GRV) dsRNAs. We were able to estimate the dsRNA content of infected leaves, identify the dsRNA species present in crude extracts and to determine the Tm- values of GRV dsRNA-3.     

Consequences of nucleic acid conformation on the binding of a trinuclear platinum drug.

BBR3464, a charged trinuclear platinum compound, is the first representative of a new class of anticancer drugs to enter phase I clinical trials. The structure of BBR3464 is characterized by two [trans-PtCl(NH(3))(2)] units linked by a tetraamine [trans-Pt(NH(3))(2)?H(2)N(CH(2))(6)NH(2)?(2)] unit. The +4 charge of BBR3464 and the separation of the platinating units indicate that the mode of DNA binding will be distinctly different from those of classical mononuclear drugs such as cisplatin, cis-[PtCl(2)(NH(3))(2)]. The reaction of BBR3464 with three different nucleic acid conformations was assessed by gel electrophoresis. Comparison of single-stranded DNA, RNA, and double-stranded DNA indicated that the reaction of BBR3464 with single-stranded DNA and RNA was faster than that with duplex DNA, and produced more drug-DNA and drug-RNA adducts. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry was used to further characterize the binding modes of BBR3464 with the DNA substrates. BBR3464 binding to different nucleic acid conformations raises the possibility that the adducts of single-stranded DNA and RNA may play a role in the different antitumor efficacies of this novel drug as compared with cisplatin.     

Interaction of trivaline with single-stranded polyribonucleotides]

Binding of tripeptide H-Val3-(NH)2-Dns (TVP) to polyribonucleotides was studied by fluorescence methods, circular and flow linear dichroism, equilibrium dialysis and electron microscopy. It was found that TVP binds to poly(U) in monomer, dimer and tetramer forms with binding constants of about 10(3), 40, 18.10(4) M, respectively. The cooperativity parameter for peptide dimer binding is 2000. The peptide forms tetramer complexes with poly(A), poly(C), poly(G) also. The formation of a complex between the peptide tetramer and nucleic acid is accompanied by a significant increase in the fluorescence intensity. The cooperative binding of TVP dimers to poly(U), poly(A), poly(C) is accompanied by a dramatic decrease in the flexibility of polynucleotide chains. However, it has a small effect (if any) on the flexibility of the poly(G) chain. The observed similarity of thermodynamic, optical and hydrodynamic++ properties of TVP complexes with single-stranded and double-stranded nucleic acids may reflect a similarity in the geometries of peptide complexes with nucleic acids. Electron microscopy studies show that peptide binding to poly(U) and dsDNA leads to compactization of the nucleic acids caused by interaction between the peptide tetramers bound to a nucleic acid. At the first stage of the compactization process the well-organized rod-like particles are formed, each consisting of one or more single-stranded polynucleotide fibers. Increasing the peptide concentration stimulates a side-by-side association and folding of the rods with the formation of macromolecular "leech-like" structures with the thickness of 20-50 nm.     

The existence of eukaryotic ribonucleoprotein consensus sequence-type RNA-binding proteins in a prokaryote, Synechococcus 6301.

A group of proteins containing a conserved ribonucleoprotein consensus sequence (RNP-CS)-type RNA-binding domain (CS-RBD) of approximately 80 amino acids is present in eukaryotic cells and binds specifically to a wide variety of RNA molecules. We have isolated 12 kDa single-stranded DNA binding proteins from the unicellular cyanobacterium Synechococcus 6301. The amino-terminal sequence was determined and two distinct genomic clones were isolated from a Synechococcus 6301 genomic library. Sequence analysis revealed that two closely related proteins contain a single CS-RBD of 82 amino acids and are named as 12RNP1 and 12RNP2. Both of the CS-RBDs share the highest amino acid identity with those of chloroplast ribonucleoproteins (40-51%). The 12RNP proteins were expressed in Escherichia coli bearing plasmids encoding glutathione S-transferase/12RNP fusion proteins and subjected to in vitro nucleic acid-binding assay. Both 12RNP1 and 12RNP2 bind to RNA homopolymers poly(U) and poly(G), indicating that they might be RNA-binding proteins. This is the first example of such proteins in prokaryotes. The 12RNP1 and 12RNP2 genes are transcribed as monocistronic mRNAs and the steady-state mRNA level of 12RNP1 is over 20-fold than that of 12RNP2. Due to the easiness of genetic manipulations the cyanobacterium will provide an excellent system to analyze the function of not only cyanobacterial but also plant RNA-binding proteins.     

A new approach to the isolation of RNA-DNA hybrids and its application to the quantitative determination of labeled tumor virus RNA

A procedure has been developed by which the hybrid formed between a labeled RNA and complementary DNA can be selectively separated from all other single and double-stranded nucleic acids. We describe the application of this procedure to the quantitative determination of labeled avian tumor virus RNA. Purified DNA complementary to avian myeloblastosis virus RNA is extended at its 3′ terminus with 40 to 60 dCMP residues, using terminal deoxynucleotidyl-transferase. The elongated DNA is annealed with the labeled nucleic acid preparation and the mixture is passed through a column of Sephadex to which poly(I) has been covalently bound. The poly(I) retains the specific RNA-DNA hybrids by virtue of their poly(C) extension. The column is washed with RNAase to degrade nonhybridized RNA, the RNA retained on the column is eluted with formamide and its radioactivity is determined. The background hybridization was reduced to 0.003 to 0.008% by addition of oligo(C)_5.20 to the hybridization mixture and by carrying out the adsorption to the poly(I)-Sephadex column in the presence of poly(U). The hybridization efficiency was about 50%. The content of radioactive Rous sarcoma virus-specific RNA was determined in infected and uninfected cells after labeling with [³H]uridine for two hours. The content of labeled virus-specific RNA in infected cells was 0.6 to 0.9% and 0.05% in uninfected cells. The value found for monkey cell RNA was 0.009%. This method can be used for the detection of hybrids between labeled RNA and complementary DNAs too short to allow quantitation by conventional methods. If the RNAase step is omitted the procedure can be used for the isolation of any RNA for which a complementary DNA is available, as well as for its precursor.     

Purification and characterization of a major endonuclease from rat liver nuclei.

A major endonuclease has been purified approximately 800-fold from rat liver nuclei using poly(A) as substrate. The enzyme had a molecular weight of about 50,000, and active fractions were obtained which contained no nucleic acid. Enzymatic activity was optimal between pH 6 and 7 and was totally dependent on the presence of a divalent cation. The reaction was inhibited by high ionic strength, polydextran sulfate, heparin, and sodium pyrophosphate. The purified enzyme readily hydrolyzed poly(A), poly(U), poly(C), and denatured DNA, whereas poly(G) was not degraded, and transfer RNA, ribosomal RNA, and native DNA were hydrolyzed only at relatively slow rates. These data suggest that the enzyme may be specific for single-stranded polynucleotides. The purified enzyme was essentially devoid of exonuclease activity, and the products of exhaustive endonuclease digestion of poly(A) were small oligonucleotides terminated with a 5'-phosphoryl group. Evidence was obtained that this endonuclease is localized in the nucleoplasm. Possible functions for this activity are discussed.     

Molecular size and shape of beta-connectin, an elastic protein of striated muscle

Connectin is an elastic protein of vertebrate striated muscle, and consists of doublet components, alpha and beta (also called titins 1 and 2). In the present study, beta-connectin isolated in the native state was investigated in order to characterize its molecular size and shape. The molecular weight was approximately 2.1 X 10(6) (SDS gel electrophoresis) or 2.7 X 10(6) (sedimentation equilibrium). The sedimentation coefficient (SO20, w) was 17S in 0.1 M phosphate buffer, pH 7.0. The intrinsic viscosity measured in an Ostwald-type viscometer was 1.8 dl/g. However, the viscosity was greatly dependent on the velocity gradient, and at a very low velocity gradient of 0.0007 s-1, a solution of connectin (0.3 mg/ml) showed a viscosity value of 17,000 cp. Flow birefringence measurements suggested a length distribution ranging from 300 to 450 nm. Electron microscopic observations revealed that connectin is a long flexible filament and the peaks of frequency of length distribution were at 150, 300, 450, and 600 nm. It was tentatively assumed that the connectin molecule is 300-400 nm long and 34-38 nm wide. It is likely that beta-connectin is derived from alpha-connectin, which has an apparent molecular weight of 2.8 X 10(6).     

NB-506, an indolocarbazole topoisomerase I inhibitor, binds preferentially to triplex DNA

A novel competition dialysis method was used to study the structural selectivity of the nucleic acid binding of NB-506, a promising indolocarbazole anticancer agent. A pronounced preference for NB-506 binding to the DNA triplex poly [dA]:(poly[dT])(2) was observed among potential binding to 12 different nucleic acid structures and sequences. Structures included in the assay ranged from single-stranded DNA, through a variety of right-handed DNA duplexes, to multistranded triplex and tetraplex forms. RNA and left-handed Z DNA were also included in the assay. The preferential binding to triplex was confirmed by UV melting experiments. The novel and unexpected structural selectivity shown by NB-506 may arise from a complementary shape between its extended aromatic ring system and the planar triplex stack.     

The determination of secondary structure in the poly(C) tract of encephalomyocarditis virus RNA with sodium bisulphite.

The degree of secondary structure in the poly(C) tract of encephalomyocarditis virus (EMCV) RNA has been investigated using sodium bisulphite, which brings about the hydrolysis of non-base-paired cytidylic acid to uridylic acid in RNA. The percentage conversion of C to U in the poly(C) region of native EMCV RNA was similar to that found in a synthetic polynucleotide lacking secondary structure [poly(C)]. When poly(I) was annealed to either native or denatured EMCV RNA, it protected the poly(C) tract from the action of bisulphite. It is concluded that the poly(C) tract of EMCV RNA in solution is very largely single-stranded.     

Isolation and properties of a single-strand 5'----3' exoribonuclease from Ehrlich ascites tumor cell nucleoli

A single-strand-specific, nucleolar exoribonuclease from Ehrlich ascites tumor cells has been isolated and purified free from other nucleases. The exonuclease degraded single-stranded RNA processively from either a 5'-hydroxyl or a 5'-phosphorylated end and released 5'-mononucleotides. The enzyme digested single-strand poly(C), poly(U), and poly(A) equally well but did not degrade duplex poly(C).poly(I) or poly(A).poly(U). Less than 0.2% of duplex DNA or 1.5% of heat-denatured DNA was degraded under the conditions which resulted in greater than 26% degradation of RNA. The ribonuclease required Mg2+ (0.2 mM) for optimum activity and was inhibited by ethylenediaminetetraacetic acid but not by human placental RNase inhibitor. The native enzyme had a Stokes radius of 42 A and a sedimentation coefficient (S20,w) of 4.3 S. From these values, an apparent molecular weight of 76 000 was derived by using the Svedberg equation. The localization and unique mode of degradation suggest a role for the 5'----3' exoribonuclease in ribosomal RNA processing.     

LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA

Locked nucleic acid (LNA) is a nucleic acid analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation. LNA oligonucleotides display unprecedented hybridization affinity toward complementary single-stranded RNA and complementary single- or double-stranded DNA. Structural studies have shown that LNA oligonucleotides induce A-type (RNA-like) duplex conformations. The wide applicability of LNA oligonucleotides for gene silencing and their use for research and diagnostic purposes are documented in a number of recent reports, some of which are described herein.