Sequential administrations of polyriboinosinic acid and polyribocytidylic acid induce interferon and depress the hepatic cytochrome P-450-dependent monooxygenase system

Double-stranded polyriboinosinic acid·polyribocytidylic acid is a potent interferon inducing agent and depressant of hepatic cytochrome P-450 monooxygenase systems. Single-stranded polyriboinosinic acid or polyribocytidylic acid are not. However, it is known that interferon is induced in mice when the administration of polyriboinosinic acid is followed shortly thereafter by the administration of polyribocytidylic acid. The current study demonstrates that this sequential administration of single-stranded polynucleotides induces serum and hepatic interferon and depresses the cytochrome P-450 systems. Neither of these effects were seen when the order of administration of these polynucleotides was reversed.     

The binding of poly(rI) - poly(rC) to human fibroblasts and the induction of interferon.

Human embryonic fibroblasts produce interferon when incubated at 37 degrees C after being treated at 4 degrees C with poly(rI) - poly(rC), either by addition of the double-stranded duplex or by sequential addition of the constitutent single-stranded polynucleotides. Cells which have been incubated with double-stranded poly(rI) - poly(rC) can be prevented from forming interferon by washing the cells with high concentrations of salt, immediately after adsorption of polynucleotides, or by incubation of the cells with single-stranded polynucleotides. The inhibition is probably due to displacement of the inducing molecule from the cell surface. Interferon production by cells treated sequentially with poly(rI) and poly(rC) is not inhibited by either of these treatments and the polynucleotides are not easily displaced from the cell surface.     

Structural requirements of polynucleotides for the activation of (2'' - 5'')An polymerase and protein kinase.

Two enzymatic pathways are involved in the inhibitory effects of double-stranded (ds)RNA on protein synthesis in cell extracts derived from interferon-treated human fibroblasts or HeLa cells, an oligonucleotide polymerase that synthesizes (2'-5')An from ATP and a protein kinase that phosphorylates the alpha subunit of initiation factor eIF-2 as well as a polypeptide of Mr = 72,000. We have now evaluated the activation of both the (2'-5')An polymerase and protein kinase by a large variety of polynucleotides, triple-stranded and synthetic dsRNAs, homopolymers, alternating copolymers, triple-stranded polymers, purine-purine duplexes and purine-pyrimidine duplexes with modifications at either the pyrimidine or ribose moieties. All these polynucleotides have been the subject of previous interferon induction studies. Some polynucleotides, i.e. (I)n.(C)n and mycophage dsRNA, which have been recognized as excellent interferon inducers, were also potent activators of both (2'-5')An polymerase and protein kinase, whereas non-inducers such as (A)n. (X)n and (A)n. (br5U)n did not activate either the kinase or the polymerase. However, some polymers like (I)n.(br5C)n, (difl)n(C)n and (dIcl)n (C)n, while potent interferon inducers and kinase activators, behaved poorly as activators of the (2'-5')An polymerase. Other polymers, i.e. (dAfl)n (U)n and (A)n.(U)nl (I)n, that do not induce interferon, activated the kinase but not the polymerase. Finally, (I)n (s2c)n, a relatively potent interferon inducer, did not activate either kinase or polymerase. These findings indicate that there is no simple relationship between the interferon-inducing ability of dsRNAs and their stimulating effects on (2'-5')An polymerase and protein kinase activity.     

Interaction of nucleic acids with electrically charged surfaces: II. Conformational changes in double-helical polynucleotides

The influence of adsorption of double-stranded (ds) DNA, ds RNA and homopolymeric pairs at a mercury electrode on conformation of these polynucleotides was studied. Changes in the polarographic reducibility of polynucleotides, which were followed by means of normal pulse polarography and linear sweep peak voltammetry at the dropping mercury electrode were exploited to indicate conformational changes. It was found that, as a consequence of adsorption of ds polynucleotides on the negatively charged electrode conformational changes similar to denaturation take place in a narrow potential region around ?1.2 V (the region U). After sufficiently long time of the contact with the electrode (under our conditions about 10 s) these changes reach limiting values, which can approach total denaturation. Upon adsorption of ds polynucleotides on the electrode charged to more positive potentials than the region U either (1) no conformational changes occur or (2) only a small part of the polynucleotide (probably labile regions of the ds molecule) is very quickly denatured - the remainder of the molecule preserves its ds structure. Conformational changes of adsorbed ds polynucleotides are influenced by factors which change the stability of ds polynucleotides in solution. It is supposed that denaturation of ds polynucleotides in the region U might result from the strains connected with the repulsion of certain segments of the molecule anchored on the electrode from the negatively charged surface.     

Control of Interferon Synthesis: Effect of Diethyl-aminoethyl-Dextran on Induction by Polyinosinic-Polycytidylic Acid

Interferon production in cultures of rabbit kidney cells (RKC) stimulated with 10 to 250 mug of polyinosinic-polycytidylic acid (poly I.poly C) per ml peaked at 3 to 4 hr after the exposure of cells to inducer and rapidly declined thereafter. On the other hand, RKC stimulated with poly I.poly C (10 or 2 mug/ml) in the presence of diethylaminoethyl (DEAE)-dextran (100 or 20 mug/ml, respectively) produced a protracted interferon response, with the release of interferon continuing for over 24 hr. The kinetics of interferon production in RKC stimulated with lower concentrations of the mixture of poly I.poly C and DEAE-dextran were similar to the response produced by poly I.poly C alone (10 to 250 mug/ml). Only the responses that terminated early were paradoxically enhanced by treatment with low doses of actinomycin D or with cycloheximide. Cells stimulated with 50 mug of poly I.poly C/ml showed hyporesponsiveness to a second interferon induction with poly I.poly C when restimulated 7 hr after primary induction. This hyporesponsiveness could be overcome by restimulating with higher concentrations of the poly I.poly C-DEAE-dextran complex. The results are compatible with the hypothesis that the early termination of interferon production and hyporesponsiveness to repeated induction with poly I.poly C are due to a cellular repressor exerting negative control on interferon synthesis, and that the increased cellular uptake of poly I.poly C in the presence of DEAE-dextran may effectively neutralize the repressor. These results also suggested that the often observed different kinetics and the varied effects of inhibitors of ribonucleic acid or protein synthesis on interferon responses in various cells and in cells stimulated with different inducers (such as with viruses as compared with polynucleotides) need not imply the existence of fundamentally different mechanisms of interferon production.     

Induction of interferon by polyribonucleotides containing thiopyrimidines.

The influence of thioketo substitution in pyrimidine bases of double-stranded polynucleotides on interferon induction was investigated. The stabilizing effect of 2-thioketo substitution was reflected in the increased interferon inducing activity of poly(A-s2U) over that of poly(A-U). Poly(A-s2U) and poly(I)-poly(s2C) were as effective as poly(I)-Poly(C) in rabbit cells. Poly(I)-poly(C) and poly(I)-poly(s2C) were compared in several animal species. No differences in biological effects were observed in rabbits and dogs. In rodents, poly(I)-poly(s2C) was less effective and less toxic.Poly(I)-poly(s2C) was highly resistant against degradation by human serum. Further investigations seem to be justified to elucidate whether this property offers any advantages for the potential clinical utilization of poly(I)-poly(s2C).     

Polynucleotide aggregates enhance the transport of protein at the surface of cultured mammalian cells.

It was known that polycationic polymers enhance the entry of macromolecules into cells. We now show that polynucleotides may have similar effects, when used as large aggregates. Poly(1-vinylcytosine):polyinosinic acid, an inducer of interferon production in human cells, can cause at 40 mug/ml a 75-fold enhancement of albumin uptake by sarcoma cells in culture. Most of this activity (85%) is related to the presence of aggregates retained by 0.65 mu millipore membranes. The prior finding that enhancers of albumin transport have increasing effects with increasing molecular sizes may thus extend to complexes of supramolecular sizes.     

Polynucleotide aggregates enhance the transport of protein at the surface of cultured mammalian cells

It was known that polycationic polymers enhance the entry of macromolecules into cells. We now show that polynucleotides may have similar effects, when used as large aggregates. Poly(1-vinylcytosine):polyinosinic acid, an inducer of interferon production in human cells, can cause at 40 μg/ml a 75-fold enhancement of albumin uptake by sarcoma cells in culture. Most of this activity (85%) is related to the presence of aggregates retained by 0.65 μ millipore membranes. The prior finding that enhancers of albumin transport have increasing effects with increasing molecular sizes may thus extend to complexes of supramolecular sizes.     

Alkyl substituent in place of the thymine methyl group controls the A-X conformational bimorphism in poly(dA-dT).

Circular dichroism studies of a family of poly(dA-y5dU) polynucleotides (y = H, methyl, ethyl, propyl, butyl or pentyl) were conducted in water-alcohol solutions containing sodium or cesium counterions. The polynucleotides denatured or adopted A- or X-DNA double helices depending on the concentration and type of alcohol, type of counterions and the length of the aliphatic substituent in place of the thymine methyl group. Short aliphatic substituents and sodium cations favored A-DNA while long aliphatic substituents and cesium cations promoted X-DNA. This study demonstrates delicacy of the conformational equilibrium of poly(dA-dT) between the A- and X-DNA double helices which depends on both intramolecular and intermolecular factors.     

银染增强的纳米金标记探针对微量核酸的检测

本研究利用银染增强的纳米金技术建立了一种简单快速的核酸定量方法.该方法基于纳米金与烷巯基修饰的寡核苷酸分子共价键合作用,将纳米微粒报告基团标记在与靶核酸一端序列互补的寡核苷酸上,同时生物素化修饰另一端互补序列.靶核酸与两段寡核苷酸探针杂交后,借亲和素固定在酶标板孔内,通过纳米金催化的银染放大效应产生高灵敏的识别信号,适时记录其吸光度值从而实现核酸分子的定量.该检测方法检测单链核酸分子的灵敏度达0.1 fM,双链分子为10 fM.     

Evidential study of correlated events in biochemistry: Physicochemical mechanisms of nucleic acid hydration as revealed by factor analysis

Using a factor analysis technique, the experimental physicochemical data on the hydration of mononucleotides, several polynucleotides, their double-helical complexes and natural DNAs were studied. The information about the factors determining the changes in physicochemical parameters vs the hydration was obtained. This work discusses a possible physical sense of the factors obtained and the expedience of using factor analysis to interpret the molecular-biophysical experiments.     

The effect of the chain length of polynucleotides on their binding with platinum complexes.

The effect of the length of polynucleotides on their binding with platinum complexes was studied. The highest reaction rate was observed in the reaction with guanosine-containing polynucleotides, whereas cytidine- and adenosine-containing polynucleotides were much less efficient. The monoaqua-forms of the platinum complexes exhibited the highest reactivity in the interaction with polynucleotides in solution. The mechanism implies the formation of the monodentate complex at the first stage which is transformed into the corresponding bidentate complex of chelate type at the second stage. Increase in the length of the polynucleotide chain was shown to enhance its interaction with the platinum complexes.     

Antigenic structure of double-stranded RNA analogues having varying activity in interferon induction.

Antibodies were induced by immunization of rabbits with methylated bovine serum albumin complexes of: poly(I).poly(BC), an effective interferon inducer; poly(c7A).poly(rT), a noninducer that can block induction by active poly(A).poly(rT); and poly(A).poly(Um), which has neither inducing nor blocking activity. Similar complexes of f2 phage RNA or tRNA did not induce anti-nucleic acid antibodies. Each anti-polynucleotide serum contained some antibodies specific for double-stranded structure. Antibodies were immunospecifically purified from precipitates made with each serum and homologous or cross-reacting double-stranded polynucleotides. The purified antibodies distinguished among varying helices bearing base or ribose modifications. Antipoly(I).poly(BC) specificity paralleled that of the interferon induction system. Anti-poly(A).poly(Um) specificity favored the 2'-modified polymers. Anti-poly(c7A).poly(rT) antibodies were the least discriminating. Cross-reaction results indicated that some antibodies reacted with determinants that included both sugar-phosphate backbones. In far antibody excess, antigen:antibody ratios in precipitating complexes reached a minimum of 7 to 12 base pairs per bivalent IgG molecule. Single antigenic determinants may span about 4 base pairs, with primary contact sites including the phosphate groups and the furanose.     

Nucleic acid vibrational circular dichroism, absorption, and linear dichroism spectra. I. A DeVoe theory approach.

Infrared (IR) vibrational circular dichroism (VCD), absorption, and linear dichroism (LD) spectra of four homopolyribonucleotides, poly(rA), poly(rG), poly(rC), and poly(rU), have been calculated, in the 1750-1550 cm-1 spectral region, using the DeVoe polarizability theory. A newly derived algorithm, which approximates the Hilbert transform of imaginaries to reals, was used in the calculations to obtain real parts of oscillator polarizabilities associated with each normal mode. The calculated spectra of the polynucleotides were compared with previously measured solution spectra. The good agreement between calculated and measured polynucleotide spectra indicates, for the first time, that the DeVoe theory is a useful means of calculating the VCD and IR absorption spectra of polynucleotides. For the first time, calculated DeVoe theory VCD and IR absorption spectra of oriented polynucleotides are presented. The calculated VCD spectra for the oriented polynucleotides are used to predict the spectra for such measurements made in the future. The calculated IR spectra for the oriented polynucleotides are useful in interpreting the linear dichroism of the polynucleotides.     

Alkyl Substituent in Place of the Thymine Methyl Group Controls the A-X Conformational Bimorphism in Poly(dA-dT)

Abstract

Circular dichroism studies of a family of poly(dA-y⁵dU) polynucleotides (y = H, methyl, ethyl, propyl, butyl or pentyl) were conducted in water-alcohol solutions containing sodium or cesium counterions. The polynucleotides denatured or adopted A- or X-DNA double helices depending on the concentration and type of alcohol, type of counterions and the length of the aliphatic substituent in place of the thymine methyl group. Short aliphatic substituents and sodium cations favored A-DNA while long aliphatic substituents and cesium cations promoted X-DNA. This study demonstrates delicacy of the conformational equilibrium of poly(dA-dT) between the A- and X-DNA double helices which depends on both intramolecular and intermolecular factors.     

Effect of the protein product of phage f1, gene 5, on heat denaturation of synthetic polynucleotides

The influence of phage f1 gene 5 protein on melting of the synthetic polynucleotides has been investigated, using UV-spectroscopy. In our experiments we have varied the proteins concentration. It has been shown, that the protein lowers the melting temperature of the studied polynucleotides (d/A--Tn dAndTn, rAndTn, rAn.r n, dAn.rn). The melting temperatures and the shapes of melting curves of various polynucleotides differ when the same protein concentrations are used. We have shown that the protein binds to the double-stranded polynucleotides, containing ribo-ribo-, deoxyribo-ribo-chains. The difference in melting temperatures and shapes of melting curves was explained using the data about the differences in the secondary structure of these polynucleotides. Only for d/A-Tn renaturation was observed after sample cooling. It may reflect the single-stranded hairpin structure of this polynucleotide.     

Antitemplate effect of polynucleotides and their hybrid complexes

In continuation of efforts to correlate the antitemplate activities of modified polynucleotides with their structure, and to understand the factors governing both their potency and stability, a group of single-stranded poly(ribo- and deoxyribo-) nucleotides, and the "hybrid" double-stranded complexes were prepared and investigated. The double-stranded hybrid poly(A,hs5U).poly(dT) section was found to be more stable to murine blood nucleases than was the single-stranded poly(A,hs5U). In a comparative study as inhibitors of the DNA polymerase alpha from rat hepatoma, the results showed that the modified polynucleotides were more potent than the unmodified ones, in general, the polydeoxyribonucleotides were better antitemplates than their ribo counterparts and the poly(A70,hs5U30).poly(dT) hybrid was more active than either of the single-stranded components. Thus it is possible to increase the nuclease resistance of the modified polyribonucleotides by forming hybrid complexes with complementary polydeoxyribonucleotides, and at the same time, to augment their antitemplate activities.     

Spin-labeled polyribonucleotides

Poly (U), poly (C) and poly (A) were spin labeled with N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)-imidazole. This spin label interacts selectively with 2' OH ribose groups of polynucleotides and does not modify the nucleic acid bases. The extent of spin labeling is not dependent upon the nature of the base and is entirely determined by rigidity of the secondary structure of the polynucleotide. The extent of modification for poly (U), poly (C) and poly (A) was 4.2, 1.7 and 1.5 per cent, respectively, the secondary structure of the polynucleotides being practically unchanged. Some physico-chemical properties of the spin-labeled polynucleotides were investigated by ESR spectroscopy. Rotational correlation times of the spin label and activation energy of its motion were calculated.     

Intrinsic Enantioselectivity of Natural Polynucleotides Modulated by Copper Ions

Natural polynucleotides including Micrococcus lysodeikticus and calf thymus DNA exhibit enantioselective recognition to S‐ofloxacin regulated by Cu²⁺. This is the first report that ofloxacin and Cu²⁺ have cooperative effects on the local distortions of polynucleotides. At the [Cu²⁺]/[base] ratio of 0.1, S‐ofloxacin is more liable to induce the locally distorted structures of polynucleotides, of which the association constant of S‐ofloxacin toward DNA‐Cu(II) is three times higher than that of the R‐enantiomer. The apparent increase of adsorption capability and cooperativity, as well as the change of adsorption mechanism were detected in the adsorption of ofloxacin enantiomers on polynucleotides upon Cu(II)‐coordination. This study not only discloses the effect of the chiral drug on the structural transition of long double‐stranded DNA, but provides fundamental data to develop a novel enantioseparation method based on natural polynucleotides. Chirality 27:306‐313, 2015. © 2015 Wiley Periodicals, Inc.