Adducts of Mannose 6-Phosphate with 5-Iodo-2′-Deoxyuridine and 2-5A as Potential Antiviral Agents

Abstract

To examine the possibility that the mannose 6-phosphate receptor system might be capitalized upon to facilitate uptake of nucleotides or nucleotides into cell, adducts of mannose 6-phosphate with 5-iodo-2′-deoxyuridine 5′-monophosphate and with adenosine 5′-monophosphate, p5′A2′p5′A and p5′A2′p5′A2′p5′A were prepared and evaluated for their antiviral activities. The adducts with 2′,5′-oligoadenylates possessed no significant antiviral activity. The adduct with 5-iodo-2′-deoxyuridine 5′-monophosphate showed activity that could be fully explained by extracellular cleavage to free 5-iodo-2′-deoxyuridine.     

Binding sites for type C viral phosphoprotein on the viral RNA genome

The distribution of binding sites for R-MuLV p12 phosphoprotein on the viral genome has been examined. Ribonucleoprotein complexes formed using 3′-poly A-containing viral RNA fragments of varying lengths and $\text{in vitro}$ radioiodinated p12 protein have been analyzed by sedimentation velocity and buoyant density gradients. Binding sites for 2–3 molecules of p12 protein can be detected within the first 400 nucleotides from the 3′-poly A segment. The possible presence of binding sites near the middle of the genome (～2500 nucleotides from the 3′-end) and very close to the 5′-terminus (within the terminal 100–200 nucleotides) is also indicated.     

Synthesis and Biological Activity of 3′-Modified 2′-5′ Adenylate Trimers

Abstract

One of the most important mediators in the mode of action of interferon is the (2′-5′)(A)n synthetase-RNase L pathway. The 2′-5′oligoadenylates (2–5A), synthesized from ATP, activate a pre-existing endonuclease that cleaves single-stranded RNA. The biological activity of 2–5A is rapidly lost due to cleavage of the 2′-5′ internucleotide bond by a specific 2′-5′-phosphodiesterase starting at the 3′end. This rapid cleavage and the poor uptake of 2–5A in intact cells limit the use of 2–5A as an antiviral or antineoplastic agent. Although several modified 2–5A analogues have been synthesized in order to improve the enzymatic stability, only few have proven to be resistant to degradation and still able to activate the 2–5A dependent endonuclease. 1-4 On the other hand, relative drastic methodology such as calcium coprecipitation, microinjection and liposome encapsulation5 has been used to introduce 2–5A into intact cells. Here, we present the synthesis and biological activity of oligoadenylates in which one or more adenosine residues were replaced by 9-(3-azido-3-deoxy-6-D-xylofuranosyl)adenine or 9-(3-amino-3-deoxy-D-xylofuranosyl)adenine. The oligonucleotides were synthesized by the phosphotriester method with triisopropylbenzenesulfonyl-chloride in the presence of N-methylimidazole as the condensing agent. The p-nitrophenylethyl group was used as the protecting group for the 2′-hydroxylfunction .(carbonate), the internucleotide linkage (phosphate ester) and the exocyclic amino groups of the heterocyclic base (carbamate). Bis(p-nitrophenylethy1)phosphoromonochloridate was used to phosphorylate the 5′-hy-droxyl group. All these blocking groups were removed with DBU in pyridine.     

The addition of 5′ cap structures occurs early in hnRNA synthesis and prematurely terminated molecules are capped

After cells were labeled by brief exposure to ³H-methyl-L-methionine, the majority of labeled 5′ terminal cap I (m⁷GpppN₁mpN₂p) oligonucleotide structures were in nuclear RNA (hnRNA) molecules ～750 nucleotides or less in length. After longer label times, the proportion of cap I structures in nuclear molecules longer than mRNA rose to approximately 60% of the total, but approximately 40% of the cap I structures were still in molecules shorter than ～750 nucleotides. The cap I structures in both long and short hnRNA chains contained all four 2′ methylated nucleotides in the N₁ position in about the same proportion as in mRNA. None of the large hnRNA molecules could be demonstrated to contain 5′ pppXp termini; the only such terminus in high molecular weight RNA was pppAp which was decreased markedly by low doses of actinomycin and is presumably the terminus of pre-rRNA. These results raise the possibilities that hnRNA chains can initiate with any of the four nucleotides, that capping occurs very close to or at the start of hnRNA chain synthesis and that approximately 40% of the hnRNA chains may be prematurely terminated.     

Regulation of Acid Phosphatase Synthesis in Baker’s Yeast Protoplast by Phosphate Compounds

The regulation of acid phosphatase synthesis by various phosphate compounds was examined in Baker’s yeast protoplasts. Synthesis was repressed by inorganic phosphate and phosphomonoesters. Phosphomonoesters were hydrolysed by a small amount of non-specific acid phosphatase present in the protoplast membrane. The inorganic phosphate that was liberated and incorporated into protoplasts probably repressed acid phosphatase synthesis. Phosphodiesters, such as 3′, 5′-cyclic AMP, 3′, 5′-cyclic CMP and 3′, 5′-cyclic GMP, promoted acid phosphatase synthesis. The effect of 3′, 5′-cyclic AMP was not to overcome hexose repression, because high hexose did not repress acid phosphatase synthesis. 3′, 5′-cyclic AMP did not overcome repression of the enzyme synthesis by inorganic phosphate. From these observations 3′, 5′-cyclic nucleotides probably had some effect on the yeast acid phosphatase-synthesizing system but the exact role of the nucleotides is obscure.     

Stereochemistry of 2′-5′ Linked Nucleic Acids: Crystal and Molecular Structure of Ammonium Adenylyl-2′, 5′-Adenosine Tetrahydrate: A Core Fragment of 2′-5′ Oligo A′s Produced by Interferon Induced Adenylate Synthetased

Abstract

The preponderance of 3′-5′ phosphodiester links in nucleic acids is well known. Albeit less prevalent, the 2′-5′ links are specifically utilised in the formation of ‘lariat’ in group II introns and in the msDNA-RNA junction in myxobacterium. As a sequel to our earlier study on cytidylyl-2′,5′-adenosine we have now obtained the crystal structure of adenylyl-2′,5′-adenosine (A2′p5′A) at atomic resolution. This dinucleoside monophosphate crystallises in the orthorhombic space group P2₁2₁2₁ with a= 7.956(3)Å, b = 12.212(3)Å and c = 36.654 (3) Å. CuKα intensity data were collected on a diffractometer. The structure was sloved by direct methods and refined by full matrix least squares methods to R = 10.8 %. The 2′ terminal adenine is in the commonly observed anti (χ₂ =?161°) conformation and the 5′ terminal base has a syn (χ₁ = 55°) conformation more often seen in purine nucleotides. A noteworthy feature of A2′p5′ A is the intranucleotide hydrogen bond between N3 and 05′ atoms of the 5′ adenine base. The two furanose rings in A2′ p5′ A show different conformations-C2′ endo, C3′ endo puckering for the 5′ and 2′ ends respectively. In this structure too there is a stacking of the purine base on the ribose 04′ just as in other 2′-5′ dinucleoside structures, a feature characteristically seen in the left handed ZDNA. In having syn, anti conformation about the glycosyl bonds, C2′ endo, C3′ endo mixed sugar puckering and N3–05′ intramolecular hydrogen bond A2′p5′ A resembles its 3′-5′ analogue and several other 2′-5′ dinucleoside monophosphate structures solved so far. Striking similarities between the 2′-5′ dinucleoside monophosphate structures suggest that the conformation of the 5′-end nucleoside dictates the conformation of the 2′ end nucleoside. Also, the 2′-5′ dimers do not favour formation of miniature classical double helical structures like the 3′-5′ dimers. It is conceivable, 2–5(A) could be using the stereochemical features of A2′p5′ A which accounts for its higher activity.     

Antagonism of 2–5 A-mediated inhibition of protein synthesis in intact cells by 2',5'-(pA)3

In vitro studies have shown that the translational inhibitory activity of 2–5 A can be blocked by the oligoribonucleotide 2',5'-(pA)₃. We have examined the effect of simultaneous introduction of inhibitor and antagonist into intact mouse cells using calcium phosphate coprecipitation. Upon introduction of 10⁻⁴ M 2',5'-(pA)₃ and 10⁻⁶ M 2–5 A, inhibition of protein synthesis was prevented. Efficiency of calcium phosphate precipitation of 2–5 A in the presence or absence of 2',5'-(pA)₃ was comparable. Introduction of 2',5'-(pA)₃ analogs showed that nucleotides which do not bind well to the 2–5 A dependent endonuclease do not prevent 2–5 A inhibitory activity. Thus, 2',5'-(pA)₃ functions as an antagonist of 2–5 A in vivo.     

Effect of 3′-end capping of aptamer with various 2′,4′-bridged nucleotides: Enzymatic post-modification toward a practical use of polyclonal aptamers

The capping of the 3′-ends of thrombin binding aptamers (TBAs) with bridged nucleotides increased the nuclease resistances and the stabilities in human serum. The binding abilities of the aptamers were not affected by the capping. The capping could be simply executed via a one step enzymatic process using 2′,4′-bridged nucleoside 5′-triphosphate and terminal deoxynucleotidyl transferase.     

Smart conferring of nuclease resistance to DNA by 3′-end protection using 2′,4′-bridged nucleoside-5′-triphosphates

Incorporation of 2′,4′-bridged nucleotides into the 3′-end of oligodeoxyribonucleotide (ODN) was examined using terminal deoxynucleotidyl transferase (TdT). The three types of 2′,4′-bridged nucleoside-5′-triphospates with different bridging structures used were incorporated efficiently into the 3′-end of DNA by TdT, although only single nucleotide incorporation was observed. Nuclease resistance was conferred on DNA, depending on the types of bridging nucleotides added.     

Accumulation of 5′ guanine nucleotides by mutants of Brevibacterium ammoniagenes

5′Xanthylic acid was efficiently converted to 5′guanine nucleotides (5′GMP, 5′GDP, and 5′GTP) without being degraded to guanine via 5′GMP by decoyinine resistant mutants of strain KY 13315 which had been isolated from Brevibacterium ammoniagenes and was practically devoid of 5′nucleotide degrading activity. The concentration of phosphate in the medium showed a profound effect on the ratio of the accumulated 5′guanine nucleotides, making it possible to direct the fermentation towards 5′GMP or 5′GTP. A direct accumulation of 5′guanine nucleotides from carbohydrate was possible by mixed cultivation of a 5′XMP accumulating strain and a 5′XMP converting mutant. A maximum concentration of 9.67 mg of 5′guanine nucleotides per ml was obtained directly from glucose in such a mixed culture.     

Effect of nucleotides on translocation of sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate into Golgi apparatus vesicles

Recent studies from this laboratory have suggested that rat-liver Golgi apparatus derived membranes contain different proteins which can translocate in vitro CMP-N-acetylneuraminic acid, GDP-fucose and adenosine 3′-phosphate 5′-phosphosulfate from an external compartment into a lumenal one. The aim of this study was to define the role of the nucleotide, sugar and sulfate moieties of sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate in translocation of these latter compounds across Golgi vesicle membranes. Indirect evidence was obtained suggesting that the nucleotide (but not sugar or sulfate) is a necessary recognition feature for binding to the Golgi membrane (measured as inhibition of translocation) but is not sufficient for overall translocation; this latter event also depends on the type of sugar. Important recognition features for inhibition of translocation of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate were found to be the type of nucleotide base (purine or pyrimidine) and the position of the phosphate group in the ribose. Thus, UMP and CMP were found to be competitive inhibitors of translocation of CMP-N-acetylneuraminic acid, while AMP did not inhibit. Structural features of the nucleotides which were less important in inhibition of translocation (and thus presumably in binding) of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate were the number of phosphate groups in the nucleotide (CDP and CMP inhibited to a similar extent), the presence of ribose or deoxyribose in the nucleotide, a replacement of hydrogen in positions 5 of pyrimidines or 8 in purines by halogens or an azido group. The sugar or sulfate did not inhibit translocation of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate into Golgi vesicles and therefore appear not to be involved in their binding to the Golgi membrane.     

Turnover of carnitine by rat tissues.

A small release of Pi from a diphenylamine-formic acid digest of DNA was detected after elimination of interpurine phosphodiester bonds was complete. Minor components in the DNA digest were identified as pyrimidine oligonucleotides which had lost one terminal phosphate. Isolated pyrimidine tracts released Pi on redigestion with the formic acid-diphenylamine reagent in amounts that increased with the number of nucleotides in the oligonucleotide taken. The oligonucleotides were also partially degraded by the formic acid-diphenylamine reagent and the degradation (2-3% of phosphodiester bonds between consecutive nucleotides) was almost independent of chain length. The cleavage was random with no preference for a phosphodiester bond flanked by particular nucleosides. This minor lack of specificity in the formic acid-diphenylamine-catalysed degradation of DNA can, however, account for the low recoveries of long pyrimidine tracts previously reported. Any analysis of pyrimidine tracts in a DNA molecule should make some correction for this small degree of degradation if exact assignments of the numbers of pyrimidine tracts are to be made.     

Dependence of Deformability of Geometries and Characteristics of Intramolecular Hydrogen Bonds in Canonical 2′-Deoxyribonucleotides on DNA Conformations

Abstract

The molecular structure and deformability (with respect to average geometry) of methyl ethers of canonical 2′-deoxyribonucleotides thymidine-5′-phosphate (mTMP), 2-deoxycytidine-5′- phosphate (mCMP), 2-deoxyadenosine-5′-phosphate (mAMP) and 2′-deoxyguanosine-5′- phosphate (mGMP) in different types of DNA have been calculated using B3LYP/cc-pvdz method. Comparison of energy at equilibrium conformations of nucleotides and conformations with torsion angles of backbone fixed to average values for different types of DNA reveals that incorporation of nucleotides to A-DNA macromolecules requires the minimum amount of deformation energy. Therefore, this type of DNA should be the least strained from viewpoint of intramolecular deformations of monomers. Modeling of environmental effects within the PCM approach reveals that the immersion of nucleotides in polar medium results in significant decrease of energy differences between anti conformers of all DNTs and syn conformers of mGMP This also leads to reduction by almost a half nucleotides' deformation energy facilitating formation of DNA macromolecule. Change of DNTs conformation causes switch between different types of intramolecular H bonds. Every type of DNA possesses unique set of intramolar hydrogen bonds in nucleotides.     

Bacterial Synthesis of Nucleotides

5′-Phosphoribosyl 5-amino-4-imidazole carboxamide was prepared by incubating 5-amino-4-imidazole carboxamide riboside and a phosphate compound with the bacteria characterized to phosphorylate at C_5′ via the phosphoryl transfer reaction. Aromatic phosphate compounds and 5′-nucleotides were able to act as the phosphate donor. This material was isolated chromatographically and its properties were studied. The other bacteria characterized to phosphorylate at C_{3′ (or 2′)} also phosphorylated a little probably at C_{3′ (or 2′)} of 5-amino-4-imidazole carboxamide riboside.

The phosphoryl interconversion between nucleotides and nucleosides was studied to be carried out via the phosphoryl transfer reaction observed in bacteria. The phosphotransferase activity of Ps. trifolii mediated reversibly the phosphoryl transfer between 5′-nucleotides and nucleosides, and its optimal pH was at around 8.5, whereas that of Prot. mirabilis did transfer the phosphoryl radical from 2′- and 3′-nucleotide to nucleoside at its optimal pH, around 5.0.

These donor- and product-isomer specificities of both bacteria were evident to be invariable, regardless of reaction pH and cultural conditions. These reactions, especially using the bacteria characterized to phosphorylate at C_5′ of nucleoside, were demonstrated to catalyze the phosphoryl interconversion between 5′-purine nucleotides and pyrimidine nucleosides or vice versa.     

Nucleotide sequence analysis of DNA. XI. The 3' terminal sequences of bacteriophage lambda and phi 80 DNA

Two procedures have been developed and applied to the determination of the 3′ terminal sequences of λ DNA and φ80 DNA. In the first procedure, each 3′ terminus was specifically labeled with a single ³²P-nucleotide. Radioactive oligonucleotides of different lengths were obtained by partial pancreatic deoxyribonuclease digestion. From the characteristic mobilities of these oligonucleotides in two dimensional fractionation systems, the 3′ terminal sequence -ACCCGCG for the r-strand and -GGTTACG for the l-strand of λ DNA have been determined. In the second procedure, approximately six nucleotides were removed from each 3′ terminus with exonuclease III, and they were replaced with radioactive nucleotides by partial repair synthesis. After enzymatic digestion and sequence analysis, the above sequences have been confirmed. The 3′ terminal sequences in φ80 DNA are identical to those in λ DNA at least up to the fifth nucleotide from the 3′ ends.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

A seed medium and a fermentation medium for nucleotide fermentations such as 5′ IMP, 5′GMP (plus GDP and GTP) and 5′AMP (plus ADP and ATP) with Brevibacterirm ammoniagenes ATCC 6872 were entirely chemically defined, with the use of a mixture of five amino acids.

As a result, the presence of Zn²⁺, Fe²⁺ and Ca²+ in addition to Mn²⁺ was found to be essential for the nucleotide fermentations. In particular, Zn²⁺ levels as well as Mn²⁺ affected nucleotide productions remarkably. Various fermentations proceeded favorably only when suboptimum levels of manganese (20~30 μg/liter) and zinc (100~200 μg/liter) were simultaneously present. This effect of trace metals was attributed to the fact that the excretion of R5P, a precursor of nucleotides, and those enzymes catalyzing reactions synthesizing nucleotides from R5P, ATP and purine bases were greatly stimulated by trace metals in cooperation with two vitamins, Ca-pantothenate and thiamine, and presumably high concentrations of phosphate and magnesium.

Furthermore, it was revealed that some metals were able to control the amounts of nucleotides accumulated when they were added to the broth during fermentation. For example, Hg²⁺ and Ag⁺ could increase the amounts of 5′GMP or 5′AMP, and decrease those of GTP and ATP.

Growth responses of Brevibacterium ammoniagenes ATCC 6872, capable of accumulating purine nucleotides, were investigated by the use of completely defined media.

1. Casamino acids required for its growth could be replaced by a mixture of l-histidine, l-homoserine, glycine, d, l-alanine and l-lysine. A completely defined medium for nucleotide productions was thus established by the use of this mixture.

2. High levels of phosphate inhibited growth markedly, and this inhibition was overcome by the simultaneous addition 1) of hign levels of Mg²⁺ and 2) of Mn²⁺, 3) pantothenate and 4) thiamine. Ca²⁺ had also a stimulatory effect on the growth. Therefore, a clear growth response to Mn²⁺ levels and the requirement of the two vitamins for growth emerged only under the conditions of high phosphate and magnesium salts. These 4 factors were found entirely the same as factors essential for nucleotide accumulations by Br. ammoniagenes.

     

Fluorescent labeling of fragments of high molecular weight RNA.

We describe a method to fluorescently label microgram quantities of high molecularweight RNA with acriflavine. The method involves hydrolyzing the RNA with HCl at pH 1.0 for 10 min to obtain segments of about 80 nucleotides. The 3′-terminal phosphate is removed from the ribose with alkaline phosphatase, and the terminal ribose is oxidized with periodate to form dialdehydes. Acriflavine is bound to the dialdehyde by the formation of a Schiff's base, and unbound acriflavine is removed by dialysis followed by chromatography on a Sephadex G-25 column eluted with phosphate buffered guanidine-HCl. Human 18 S rRNA bound 0.94 acriflavine molecules per 100 nucleotides and had a fluorescence excitation maximum at 460 nm and an emission maximum at 508 nm. If the hydrolysis step was omitted, this RNA bound only 0.12 acriflavine molecule per 100 nucleotides. Acriflavine-labeled high molecular weight yeast RNA showed a fluorescent intensity which was proportional to RNA concentration to a 1000-fold dilution.     

Regulation of the antiviral and anticellular activities of interferon by exogenous double-stranded RNA

The effects of double-stranded RNA (dsRNA) on interferon (IFN)-induced antiviral and anticellular activities was investigated by introducing poly(I)-poly(C) into mouse L-cells. Coprecipitation of dsRNA with calcium phosphate enabled its efficient penetration into cells in culture. Rate of cellular protein synthesis was inhibited by dsRNA only in cultures pretreated with IFN. Moreover, the anticellular effect of IFN, as measured by the inhibition of cell DNA synthesis, was also enhanced by dsRNA. The kinetics of dsRNA-mediated inhibition of protein synthesis were relatively slow as compared with the inhibitory effect of 2'-5' oligoadenylic acid (2'5'A), which was also introduced into cells by the calcium phosphate coprecipitation technique. To analyze the effects of dsRNA on the antiviral state induced by IFN, vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMC), replications were followed by measuring viral-specific RNA synthesis in the cell. Introduction of dsRNA after the infection had no effect on VSV and EMC replication in control cells, and it enhanced, to a small extent, the antiviral state of cells pretreated with IFN. In contrast, introduction of 2'5'A into virus-infected cells inhibited VSV and EMC replications regardless of IFN pretreatment. This work demonstrated that the role of dsRNA in regulating the antiviral and anticellular activities of IFN could be studied by introducing exogenous dsRNA into cells in culture by the calcium phosphate coprecipitation technique.     

Effect of guanine nucleotides on the assembly of brain microtubles: ability of 5'-guanylyl imidodiphosphate to replace GTB in promoting the polymerization of microtubules in vitro.

Polymerization of microtubule requires the presence of GTP, and the tubulin-bound GTP is hydrolyzed during microtubule formation. However, it was found that an unhydrolyzable analog of GTP, 5′-guanylyl imidodiphosphate (Gpp(NH)p), was able to replace GTP. The hydrolysis of the terminal phosphate group of GTP, therefore, does not seem to be a prerequisite to $\text{in vitro}$ assembly of microtubules. The microtubules formed in the presence of Gpp(NH)p were indistinguishable from those formed in the presence of GTP under electron microscopy, but a remarkable decrease was noted in their sensitivity to depolymerization by calcium ions.     

Phage particle-mediated gene transfer to cultured mammalian cells

Recombinant phage particles carrying the thymidine kinase (TK) gene of herpes simplex virus type 1, coprecipitated with calcium phosphate, efficiently transformed mouse Ltk- cells to the TK+ phenotype. The conditions necessary to achieve high efficiency of transfer of the TK gene by phage particle-mediated gene transfer were investigated. Of the parameters examined, the pH of the buffer used for coprecipitation of phage particles with calcium phosphate, the length of time of coprecipitation, and the length of the adsorption period were found to alter the transfer efficiency significantly. The optimal pH was 6.87 at 25 degrees C. The other optimal values for these parameters were as follows: coprecipitation time, 7 to 20 min; adsorption time, 18 to 30 h. Treatment with dimethyl sulfoxide, glycerol, or sucrose did not enhance gene transfer. The optimal conditions yielded about 1 transformant per 10(5) phage particles per 10(6) cells without carrier DNA. An increase in the dosage of phage particles, up to at least 5 x 10(7) phage particles per 100-mm dish, resulted in a linear increase in the number of transformants. Addition of carrier phage, up to 10(10) phage particles per dish, did not significantly affect the number of transformants.