Synthetic Double-stranded Polynucleotides Research Articles

Spectroscopic Study of the Binding of Netropsin and Hoechst 33258 to Nucleic Acids

The interaction of groove binding compounds — peptide antibiotic (polyamide) netropsin and fluorescent dye (bisbenzimidazole) Hoechst 33258 — with the double-stranded DNA and synthetic double-stranded polynucleotide poly(rA)-poly(rU) has been studied by spectrophotometry. Absorption spectra of these ligand complexes with nucleic acids have been obtained. Spectral changes at the complexation of individual ligands with the mentioned nucleic acids reveal the similarity of binding of each of these ligands with both DNA and RNA. Based on the spectroscopic measurements, the binding parameters of netropsin and Hoechst 33258 binding to DNA and poly(rA)-poly(rU) – K and n, as well as the thermodynamic parameters ΔS, ΔG, and ΔH have been determined. It was found that the binding of Hoechst 33258 to both nucleic acids is accompanied by a positive change in enthalpy, while in the case of netropsin the change in enthalpy is negative. Moreover, the contribution of entropy to the formation of the complexes is more pronounced in the case of Hoechst 33258.

Cyclic voltammetry of submicrogram quantities of supercoiled, linear and denatured DNAs with DNA-modified mercury electrode.

We have previously shown that a DNA-modified electrode can be prepared by immersing the hanging mercury drop electrode (HMDE) in a small volume (about 5-10 microliters) of a DNA solution. Within a short period of time the DNA is irreversibly adsorbed at the electrode, resisting subsequent washing. The electrode is then transferred into a voltammetric cell filled with the background electrolyte (that does not contain any nucleic acid) in which the voltammetric measurements are performed. This procedure is called adsorptive transfer stripping voltammetry (AdTSV). In this paper AdTS cyclic voltammetry (CV) peaks of DNA were measured to report on the stability of the attachment of plasmid DNA molecules to the electrode surface. It was shown that the attachment of plasmid DNA to the electrode was, like the case of calf thymus DNA, sufficiently stable. If the DNA-modified electrode was immersed in a protein solution no significant exchange between the DNA and protein was observed. Submicrogram amounts of DNA were sufficient to attain full coverage of the electrode at relatively short waiting times and the detection limit of the denatured DNA was below 2 ng. The intensity of the AdTS CV signals of supercoiled, linearized and thermally denatured linear DNAs differed from one another. It was shown that alkaline denaturation of linear and supercoiled DNA in solution can be studied using AdTS CV technique. It was further shown that at neutral pH the linearized plasmid undergoes denaturation due to a prolonged contact with the electrode charged to potentials around -1.2 V. Such a surface denaturation has been previously observed with calf thymus DNA and synthetic double-stranded polynucleotides. Our results show that in contrast to linear DNA, supercoiled DNA was not significantly denatured as a result of its prolonged contact with the electrode charged to various potentials in the range between -0.1 to -1.5 V.

Chemically induced viral resistance in single preimplantation bovine embryos

Viral mRNA encoding vesicular stomatitis virus glycoprotein sequences was detected and quantitated using a DNA-hybridization dot-blot technique. This assay was employed to determine if the synthetic double-stranded polynucleotide complex of polyriboinosinic-polyribocytidylic acid would elicit viral resistance in vitro in single day 9 bovine embryos. The levels of viral mRNA were assayed in 4 groups of bovine embryos: unexposed, virus-exposed, polynucleotide-treated, and virus plus polynucleotide treated. Reduced quantities of viral mRNA in single polynucleotide treated embryos demonstrated that resistance to viral infection was induced in day 9 bovine embryos.

A type II DNA-binding protein genetically engineered for fluorescence spectroscopy: the "arm" of transcription factor 1 binds in the DNA grooves.

We examined the fluorescence properties of a mutant TF1 protein (transcription factor 1; a member of the type II class of DNA-binding proteins, DBPII) containing tryptophan in place of phenylalanine (TF1-W61) at position 61 in the "arms" of the protein dimer. The time-resolved fluorescence (excited at 295 nm) of Trp61 decays as a double exponential with lifetimes and amplitudes that are comparable to those found in other tryptophan-containing proteins and peptides, and the time-resolved fluorescence polarization decay indicates that the tryptophan residue possesses considerable internal flexibility, in agreement with crystal studies of the homologous HU protein. The tryptophan emission is quenched when TF1-W61 binds to DNA, and equilibrium studies based on fluorescence show that the nonspecific binding affinity of the TF1-W61 mutant to DNA is similar to that of wild-type TF1. Comparison of the time-resolved fluorescence decay and steady-state fluorescence intensity reveals at least two general classes of Trp61 in the DNA complexes. One class of tryptophans is partially quenched, and the extent of quenching in the complexes with various natural DNAs and synthetic double-stranded polynucleotides correlates with the spectral overlap between tryptophan emission and DNA absorption, indicating that through-space excitation energy transfer contributes to the observed quenching. Comparisons between experimentally determined energy transfer rates and model calculations suggest that the Trp61 is located in one of the DNA grooves at a distance of less than 7.5 A from the DNA helix axis. The second class of Trp61 is "totally" quenched, and we attribute this to tryptophan residues that are in direct contact with the DNA bases.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of natural killer cytotoxicity by long-term treatment with double-stranded polynucleotides without induction of hyporesponsiveness.

Treatment of Wistar/AG rats with a single i.p. injection of 1 mg/kg of synthetic double-stranded polynucleotides, either polyadenylic-polyuridylic acid (rAn.rUn), or a mismatched analogue of polyinosinic-polycytidylic acid (rIn.r(C12U)n), enhanced the cytotoxicity of natural killer (NK) cells among peripheral blood leukocytes and lung intracapillary leukocytes (LICL). The enhancement reached a peak 24 h after treatment and returned to control values after 4 days. In rats given repeated injections of double-stranded polynucleotides (2 per week), the NK cytotoxicity expressed by LICL reached more than ten times (in lytic units) the control levels between day 8, after 3 injections, and day 360, after 100 injections. No hyporesponsiveness was observed. Moreover, NK activity was frequently and significantly higher in rats given multiple injections than in those given a single injection. In rats with experimentally induced P77 lung fibrohistiocytoma colonies, repeated injections of rIn.r(C12U)n stimulated NK activity and reduced the number of metastatic nodules from 172 to 19. The same significant reduction (from 172 to 27) was also observed in animals given repeated injections of rAn.rUn. However, with two models of spontaneous metastases, significant reduction in lung metastases (M37 bronchioloalveolar carcinoma) or lack of effect (S4T19 rhabdomyosarcoma) were observed.

Potentiation of the Cellular Immune Response by Adjuvants: A Limited Role for Adjuvant Induced Interferon

Adjuvants differ in capacity to induce interferon (IFN). As a consequence IFN induction by adjuvants may influence their effectiveness in enhancement of delayed type hypersensitivity (DH) reactions. In this study, the lipophilic amine dimethyl dioctadecyl ammonium bromide (DDA), the synthetic double-stranded polynucleotide polyinosinic polycytidylic acid (poly I:C), liposomes and the polyols L 101 and L 121 were compared in BALB/c mice as inducers of IFN and also as adjuvants for DH to both lysozyme and inactivated Semliki Forest virus (SFV). The antigens were injected intracutaneously, alone or mixed with adjuvant. At day 6 after the immunization, DH was elicited and measured 24 h later as increase in footpad thickness. Negatively charged liposomes and polyol L 121 were unable to enhance DH to SFV and also lack the capacity to induce IFN. Polyol L 101 induced low levels of IFN and showed only slight adjuvanticity for DH to SFV. In contrast, DDA, a moderate IFN inducer, was shown to be a very effective adjuvant for induction of DH against both lysozyme and SFV. The excellent IFN inducer, poly I:C, at the tested dose range (0.03-3.0 mg/kg), displayed only a relatively weak adjuvant activity. But low doses of poly I:C (0.03 and 0.1 mg/kg) showed still adjuvanticity in contrast to equal doses of DDA. This might be related to sufficient induction of IFN by low doses of poly I:C but not by DDA. The discrepancy observed in the relation between IFN induction and a maximal DH induction suggests that IFN is not the only factor which enhances the effectiveness of adjuvants in induction of DH.(ABSTRACT TRUNCATED AT 250 WORDS)

Liquid-crystal microphases of low-molecular weight double stranded nucleic acids and synthetic polynucleotides

A study has been made of the properties of microphases formed as a result of phase separation occurring during the mixing of water-salt solutions of native double-stranded DNA, and of synthetic double-stranded polynucleotides, with water-salt solutions of polyethylene glycol. At a definite concentration of polyethylene glycol it was found that molecules of low-molecular weight DNAs or of synthetic polynucleotides, may be in the liquid-crystal state. An intense ( Δϵ 270 ∼ 100 units) band in the circular dichroism spectrum accompanying the appearance of liquid-crystal microphase points to the existence of long-range helical order in the arrangement of nitrous bases in the composition of DNA molecules or of polynucleotides forming liquid-crystal microphases. It is shown that, irrespective of the direction of twist of polynucleotide double helices are two groups of liquid-crystal microphases differing as to the sign of intense bands in the circular dichroism spectra: liquid-crystal microphases belonging to the first group are characterized by an intense negative band, compared with an intense positive band of those belonging to the second group.

Homeostasis of the antibody response: immunoregulation by NK cells.

When injected into mice, the synthetic double-stranded polynucleotide poly(inosinic) X poly(cytidylic) acid induces high natural killer (NK) cell activity within 4 to 12 hours. Induction of NK activity in mice immunized 2 or 3 days previously, or the addition of NK cells to cultures immunized in vitro 2 or 3 days previously, promotes early termination of the ongoing primary immunoglobulin M antibody response. A target for NK cells is a population of accessory cells that has interacted with antigen and is necessary for sustaining the antibody response. The inference is strong that NK cells induced normally by immunization also terminate the usual antibody response in vivo by elimination of antigen-exposed accessory cells.

The interaction of ethidium with synthetic double-stranded polynucleotides at low ionic strength.

The interaction of ethidium with synthetic DNA and RNA double-stranded polymers at 0.01 M ionic strength, pH 7.0, has been studied by fluorimetry at low drug to nucleotide ratios. Binding constants have been calculated assuming an excluded-neighbouring site model for the interaction of ethidium with double-stranded polymers. The values obtained are poly d(AT).poly d(AT), 9.5 X 10(6) M-1; poly dA.poly dT, 6.5 X 10(5) M-1; poly d(GC).poly d(GC), 9.9 X 10(6) M-1; poly dG,poly dC, 4.5 X 1-(6) M-1; poly d(AC); poly d(GT), 9.8 X 10(6) M-1; poly d(AG).poly d(CT), 1.3 X 10(6) M-1; poly rA.poly rU, 4.1 X 10(7) M-1. The displacement of ethidium from poly d(AT).poly d(AT) by 9-aminoacridine and an acridine-containing antitumor agent (NSC 156303; 4'-(9-acridinylamino)methanesulphon-m-anisidide) has also been examined.

Depression of hepatic cytochrome P-450-dependent monooxygenase systems with administered interferon inducing agents

Cytochrome P-450-dependent monooxygenase activities and cytochrome P-450 levels were depressed in hepatic microsomes from rats treated with 12 interferon inducing agents of various types: small molecules (e.g. tilorone), an RNA virus (Mengo), a fungal mycophage (statolon), liver RNA, a synthetic double-stranded polynucleotide (poly rI · poly rC), a bacterial lipopolysaccharide ( E. coli endotoxin) and an attenuated bacteria ( B. pertussis vaccine). The results suggest that the depression of hepatic cytochrome P-450-dependent monooxygenase systems may be a general property of interferon inducing agents.

Effect of synthetic double-stranded polynucleotides on leukocyte migration

The effect of synthetic double-stranded polynucleotides (polyI:polyC) on migration of peripheral blood leukocytes from donors and patients with rheumatic fever was investigated. PolyI:polyC in large quantities was found not to inhibit migration of the donors' leukocytes, but it inhibited migration of leukocytes from patients with rheumatic fever, in whom antibodies against double-stranded RNA were found more often than in donors.

Interferon Production and Resistance to Viral Infection Induced by Poly I· Poly C in Chick Embryo Cells. II. Lack of Enhancement by DEAE-Dextran

Japanese Journal of MicrobiologyVolume 19, Issue 1 p. 65-68 NoteFree Access Interferon Production and Resistance to Viral Infection Induced by Poly I· Poly C in Chick Embryo Cells. II. Lack of Enhancement by DEAE-Dextran Haruhiko Machida, Corresponding Author Haruhiko Machida Research Laboratory, Yamasa Shoyu Co., Ltd., Choshi, ChibaRequests for reprints should be addressed to Dr. Haruhiko Machida, Research Laboratory, Yamasa Shoyu Co., Ltd., Araoi 2–550, Choshi-shi, Chiba 288, Japan.Search for more papers by this authorAkira Kuninaka, Akira Kuninaka Research Laboratory, Yamasa Shoyu Co., Ltd., Choshi, ChibaSearch for more papers by this authorHiroshi Yoshino, Hiroshi Yoshino Research Laboratory, Yamasa Shoyu Co., Ltd., Choshi, ChibaSearch for more papers by this author Haruhiko Machida, Corresponding Author Haruhiko Machida Research Laboratory, Yamasa Shoyu Co., Ltd., Choshi, ChibaRequests for reprints should be addressed to Dr. Haruhiko Machida, Research Laboratory, Yamasa Shoyu Co., Ltd., Araoi 2–550, Choshi-shi, Chiba 288, Japan.Search for more papers by this authorAkira Kuninaka, Akira Kuninaka Research Laboratory, Yamasa Shoyu Co., Ltd., Choshi, ChibaSearch for more papers by this authorHiroshi Yoshino, Hiroshi Yoshino Research Laboratory, Yamasa Shoyu Co., Ltd., Choshi, ChibaSearch for more papers by this author First published: January 1975 https://doi.org/10.1111/j.1348-0421.1975.tb00848.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat REFERENCES 1Colby, C., and Chamberlin, M.J. 1969. The specificity of interferon induction in chick embryo cells by helical RNA. Proc. Nat. Acad. Sci. U.S. 63: 160– 167. 2Dianzani, F., Baron, S., Buckler, C.E., and Levy, H.B. 1971. Mechanisms of DEAE-dextran enhancement of polynucleotide induction of interferon. Proc. Soc. Exp. Biol. Med. 136: 1111– 1114. 3Dianzani, F., Cantagalli, P., Gagnoni, S., and Rita, G. 1968. Effect of DEAE-dextran on production of interferon induced by synthetic double-stranded RNA in L cell cultures. Proc. Soc. Exp. Biol. Med. 128: 708– 710. 4Dianzani, F., Gagnoni, S., and Cantagalli, P. 1970. Studies on the potentiating effect of DEAE-dextran on interferon production induced by double stranded synthetic polynucleotides. Ann. New York Acad. Sci. 173: 727– 735. 5Long, W.F. 1971. Effect of metabolic inhibitors on viral and polynucleotide induction of chick cell interferon. Microbios 4: 253– 259. 6Long, W.F., and Burke, D.C. 1971. Interferon production by double-stranded RNA: A comparison of induction by reovirus to that by a synthetic double-stranded polynucleotide. J. Gen. Virol. 12: 1– 11. 7Machida, H., Kuninaka, A., and Yoshino, H. 1974. Interferon production and resistance to viral infection induced by poly I· poly C in chick embryo cells. I. Effect of growth medium. Japan. J. Microbiol. 18: 427– 432. 8Nordlund, J.J., Wolff, S.M., and Levy, H.B. 1970. Inhibition of biologic activity of poly I: poly C by human plasma. Proc. Soc. Exp. Biol. Med. 133: 439– 444. 9Novokhatsky, A.S., and Ershov, F.I. 1972. The effect of diethylaminoethyl dextran on the antiviral activity of the complex of polyinosinic and polycytidylic acids in tissue culture. Vopr. Virusol. 17: 312– 317. 10Pitha, P.M., and Carter, W.A. 1971. The DEAE dextran : polyriboinosinate-polyribocytidylate complex: Physical properties and interferon induction. Virology 45: 777– 781. 11Stern, R. 1970. A nuclease from animal serum which hydrolyzes double-stranded RNA. Biochem. Biophys. Res. Commun. 41: 608– 614. 12Tilles, J.G. 1970. Diethylaminoethyl-dextran enhancement of interferon induction by a complexed polyribonucleotide. Proc. Soc. Exp. Biol. Med. 133: 1334– 1341. 13Vilček, J., Barmak, S.L., and Havell, E.A. 1972. Control of interferon synthesis: Effect of diethylaminoethyl-dextran on induction by polyinosinic-polycytidylic acid. J. Virol. 10: 614– 621. Citing Literature Volume19, Issue1January 1975Pages 65-68 ReferencesRelatedInformation

In vitro studies on mouse interferons induced with phage double-stranded RNA, synthetic double-stranded polynucleotides and viral inducers.

Highly purified and concentrated interferons obtained from L cells or from mouse peritoneal leukocytes (MPL) after induction with different inducers (Newcastle disease virus, double-stranded RNAs) were similar in their electrophoretic features. Incorporation of35S-labeled methionine during induction and production of interferons was similar in both cultures. However, differences were seen in electrophoretic profiles of interferons obtained from MPL cultures at different time intervals after induction.

Specific murine B-cell activation by synthetic single-and double-stranded polynucleotides.

The synthetic single- and double-stranded polynucleotides, poly I, poly C, and poly I.C, were shown to induce thymidine incorporation in six inbred strains of murine spleen cells. This stimulation was shown to be secondary to B-cell activation and not due to contamination of the polynucleotides with bacterial lipopolysaccharide (LPS). The ability of poly I.C to act as a B-cell mitogen, in addition to its behavior as a thymic-independent antigen, suggested that these two phenomena may be related. The similarity of the molecular structure of poly I.C to LPS, a material which also acts as a thymic-independent antigen and a B-cell mitogen, supports the hypothesis that the polyvalent nature of these materials accounts for their functional interaction with murine B cells.

Coxsackievirus myocarditis: prophylaxis and therapy with an interferon stimulator.

SummaryA single dose of the synthetic double-stranded polynucleotide poly I·C inoculated intraperitoneally into mice 12 to 48 hr before challenge with coxsackievirus B-3 resulted in almost complete protection from virus-induced myocarditis. Protection was related to the presence of high titers of circulating IF in the serum. Significant protection was also obtained even when poly I·C was given 24 hr after challenge with coxsackievirus.

Enzymatic Synthesis of Deoxyribonucleic Acid: XXXVI. A PROOFREADING FUNCTION FOR THE 3′ → 5′ EXONUCLEASE ACTIVITY IN DEOXYRIBONUCLEIC ACID POLYMERASES

Abstract The Escherichia coli and T4 DNA polymerases do not extend chains in which the 3'-terminal nucleotide (primer terminus) is not paired with the template. By using synthetic double-stranded polynucleotides, the 3' → 5' exonuclease function of these polymerases was shown to be directed specifically against a mispaired or unpaired primer terminus. Chain extension of such termini begins only after all mispaired nucleotides have been removed and a basepaired terminus is reached. The latter is completely conserved while polymerization is maintained. These results suggest that the function of the 3' → 5' exonuclease activity of DNA polymerases is to remove mispaired nucleotides which have been incorrectly incorporated, thereby increasing the fidelity of template copying. The function of other E. coli exonucleases suggested by their specificities on polynucleotide substrates are trimming loose ends of DNA for exonuclease I and enlarging nicks and gaps within helical regions for exonuclease III.

Interferon Production by Double-stranded RNA: A Comparison of Induction by Reovirus to that by a Synthetic Double-stranded Polynucleotide

SUMMARY Treatment of chick cells with reovirus resulted in the production of an inhibitor of virus replication which was characterized as an interferon. No infectious virus was released, nor could any virus-specific RNA synthesis be detected in cells receiving virus. The ability of reovirus to induce interferon formation in chick cells was less sensitive to irradiation with ultraviolet light than was its ability to produce infectious virus in L cells. These data suggest that the interferon was induced by the double-stranded RNA of the inoculum virus. Comparison of this system with that induced by a synthetic double-stranded polynucleotide showed that the latter was considerably less sensitive to the effects of metabolic inhibitors, suggesting that the two inducers do not stimulate interferon formation by the same mechanism. The effect of addition of two metabolic inhibitors simultaneously to cells previously treated with a synthetic polynucleotide suggests that their effect is not a direct one on the formation of an interferon messenger RNA and its translation.

Influence of inhibitors of protein synthesis on interferon formation in mice: II. Comparison of effects of glutarimide antibiotics and tenuazonic acid

Additional information has been obtained about the different patterns of interferon synthesis or release in mice injected with virus or bacterial endotoxin. Blockade of protein synthesis at different times after injection of Newcastle disease virus effectively halted further increase of circulating interferon. Data obtained by this method showed that interferon synthesis induced by virus is continuous for at least 10–12 hours. In the case of endotoxin, an enhanced and prolonged release of interferon was seen when blockade of protein synthesis was produced up to 30 min after the injection of endotoxin. By 1 or 2 hours after the injection of endotoxin, no significant enhancement of interferon release was effected by blockade of protein synthesis, indicating that the critical events involved in the release of preformed interferon by endotoxin occur within 30 min after the stimulus is injected. When a synthetic double-stranded polynucleotide, poly rI:rC, was used as the stimulus instead of endotoxin, the temporal relationship between blockade of protein synthesis and enhancement of interferon release in mice was similar. The effects of blockade of protein synthesis in mice described above were obtained using a series of glutarimide antibiotics (cycloheximide, acetoxycycloheximide, streptovitacin A, streptimidone) as well as tenuazonic acid, a chemically unrelated antibiotic.

Relative sensitivities of viruses to different species of interferon.

Some viruses were found to be more sensitive than others to the action of interferons from certain species of animals but less sensitive to interferons from other species. Vaccinia virus was the most sensitive to mouse and hamster interferons of five viruses tested, but the least sensitive of these five viruses to human, rabbit, and bat interferons. The relative sensitivities of the viruses to interferons were found to be characteristic for each of the species tested, with those closely related phylogenetically exhibiting similar patterns of relative interferon-induced virus resistance. The amount of synthetic double-stranded polynucleotide polyinosinic acid-polycytidylic acid required to induce resistance to each of the viruses in each of the cell species correlated with the interferon sensitivities of the viruses.

Induction of interferon synthesis by synthetic double-stranded polynucleotides.

Induction of interferon synthesis by synthetic double-stranded polynucleotides.