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.     

Interferon induction by mismatched analogues of polyinosinic acid . polycytidylic acid [(Ix,U)n.(C)n].

Interruption of the (I)n strand of (I)n.(C)n by unpaired bases [(U)] yielded mismatched analogues, (Ix,U)n.(C)n which were still effective as inducers of interferon, provided the I:U ratio (x) was equal or greater than 10. In highly sensitive interferon-induction systems such as primary rabbit kidney cells and human skin fibroblasts superinduced with cycloheximide and actinomycin D, (I10,U)n.(C)n and (I50,U)n.(C)n proved nearly as active as (I)n.(C)n. By virtue of their increased susceptibility to degradation by nucleases, (Ix,U)n.(C)n complexes with 10 less than or equal to x less than or equal to 50 may be expected not to persist as long in biological fluids as (I)n.(C)n, hence to induce fewer side effects.     

Enhancement of the antiviral and interferon-inducing activities of poly r(A-U) by carminic acid

Experiments have been designed to systematically examine the effects of carminic acid (CAR) on the antiviral/interferon-inducing activity of poly r(A-U), using the human foreskin fibroblast-vesicular stomatitis virus bioassay system. Modulation of the antiviral/interferon-inducing activity of poly r(A-U) by carminic acid was examined at fixed poly r(A-U) concentrations of 0.05 mM or 0.2 mM while varying the carminic acid concentrations to produce variable CAR/ribonucleotide ratios ranging from 1:16 to 2:1. Carminic acid and poly r(A-U) were tested individually at the concentrations employed in the CAR/poly r(A-U) combinations. Neither the carminic acid alone nor poly r(A-U) alone were effective antiviral agents/interferon inducers. The antiviral/interferon-inducing activity of poly r(A-U) was potentiated twelve-fold at CAR/ribonucleotide ratios in the region of 1/6 to 1/4. These results suggest a synergism between the poly r(A-U) and the carminic acid at the concentrations employed in this study.     

Affinity chromatography on immobilised nucleotides. The synthesis, specificity and applications of immobilised inosine 5''-monophosphate

In addition to the 2'-azido analogue of (I)n x (C)n, (dIn3)n x (C)n, we have found two other (I)n x (C)n analogues, (dIfl)n x (C)n and (dIcl)n x (C)n, in which the 2'-hydroxyls of the (I)n strand are replaced by either fluorine or chlorine, to be highly effective in inducing interferon. This contrasted with the lack of interferon-inducing activity noted for various other 2'-halogeno analogues of (I)n x (C)n and (A)n x (U)n, i.e. (I)n x (dCcl)n, (dAfl)n x (U)n, (dAcl)n x (U)n, (A)n x (dUfl)n and (A)n x (dUcl)n. In most assay systems, viz. primary rabbit kidney cells, human diploid fibroblasts, HeLa cells, interferon-primed mouse L-929 cells, and intact rabbits, (dIfl)n x (C)n and (dIcl)n x (C)n induced interferon levels that were comparable to those induced by (I)n x (C)n. There was one particular system (L-929 cells treated with DEAE-dextran), however, in which (dIfl)n x (C)n and (dIcl)n x (C)n, unlike (I)n x (C)n, failed to stimulate interferon production. As monitored by both radiochemical and biological means, (dIfl)n x (C)n and, to a lesser extent, (dIcl)n x (C)n were more resistant to degradation by ribonuclease A, T1 and human serum nucleases than was (I)n x (C)n. In their reactivity towards antibodies to double-stranded RNA (dIfl)n x (C)n and (dIcl)n x (C)n conformed more closely to (I)n x (C)n than did other 2'-substituted (e.g. 2'-O-methyl or 2'-O-ethyl) analogues of (I)n x (C)n. The high interferon-inducing potency of (dIfl)n x (C)n and (dIcl)n x (C)n has both theoretical and practical implications. While our findings suggest that (dIfl)n x (C)n and (dIcl)n x (C)n should be further explored for their therapeutic potentials, they also strengthen the notion that the interferon-inducing capacity, and possibly other biological functions of double-stranded RNAs is dependent on the recognition of the overall conformation of the polynucleotide rather than on the binding of specific functional groups such as the 2'-hydroxyl group.     

Kinetic analysis of the annealing period in the formation of the poly(A)·2poly(U) triple helix

The slow kinetics of annealing processes in multistranded nucleic acids is spectrophotometrically investigated using poly(A)·2poly(U) as a model system. The absorbance changes at specific wavelengths show that double-helical (A·U) base pairs appear as transient intermediates. The annealing process is identified by the enlargement of triple-helical sequences at the cost of (A·U) base pairs and unpaired (U) residues. A large time range in the reorganization of mismatched chain configurations is characterized by a logarithmic dependence on time. This observation is quantitatively described by a kinetic model developed by Jackson. In Jackson's model the rate-limiting process in the slow annealing phase of maximizing triple-helical sequences, is the removal of strand entanglements, knots, and hairpin loops by complete unwinding of those helical stretches which stabilize the mismatched configurations. The results of the present study are briefly discussed in terms of optimum conditions for hybridization experiments and for the preparation of polynucleotide complexes commonly used to produce interferons.     

Polynucleotides. XLIV. Synthesis and properties of poly (2-azaadenylic acid) and poly(2-azainosinic acid).

Chemically synthesized 2-azaadenosine 5'-diphosphate (n2ADP) and 2-azainosine 5'-diphosphate (n2IDP) were polymerized to yield poly(2-azaadenylic acid), poly(n2A), and poly(2-azainosinic acid), poly(n2I), using Escherichia coli polynucleotide phosphorylase. In neutral solution, poly(n2A) and poly(n2I) had hypochromicities of 32 and 5.5%, respectively. Poly(n2A) formed an ordered structure, which had a melting temperature (Rm) of 20 degrees C at 0.15 M salt concentration. Upon mixing with poly(U), poly(n2A) formed a 1 : 2 complex with Tm of 41 degrees C at 0.15 M salt concentration. Poly(n2A) and poly(n2I) formed three-stranded complexes with poly(I), and poly(A), respectively. Poly(n2A) . 2poly(I), poly(A) . 2poly(n2I), and poly(n2A) . 2poly(n2I) complexes had Tm values of 23, 48, and 31 degrees C at 0.15 M salt concentration, respectively. Poly(n2I) formed a double-stranded complex with poly(C), but its Tm was very low.     

Polynucleotides. XLV Synthesis and properties of poly(2''-azido-2''-deoxyinosinic acid).

Poly (2'-azido-2'-deoxyinosinic acid), [poly (Iz)], was synthesized from 2'-azido-2'-deoxyinosine diphosphate by the action of polynucleotide phosphorylase. Poly (Iz) has UV absorption properties similar to poly (I) and hypochromicity of 11% at 0.15M Na+ and neutrality. In solutions of high Na+ ion concentration, poly (Iz) forms a multi-stranded complex and its Tm at 1.0M Na+ ion concentration was 43 degrees. Upon mixing with poly (C), poly (Iz) forms a 1:1 complex having a Tm lower than that of poly (I)-poly (C) complex in the same conditions. The effect of substitution at the 2'-position of the poly (I) strand was discussed in relation to the interferon-inducing activity.     

Synthesis and self-assembly of well-defined poly(amino acid) end-capped poly(ethylene glycol) and poly(2-methyl-2-oxazoline)

Poly(ethylene glycol) (PEG) and poly(2-methyl-2-oxazoline) (PMOx) are water-soluble, biocompatible polymers with stealth hemolytic activities. Poly(amino acid) (PAA) end-capped PEG and PMOx were prepared using amino-terminated derivatives of PEG and PMOx as macroinitiators for the ring-opening polymerization of γ-benzyl protected l-glutamate N-carboxyanhydride and S-benzyloxycarbonyl protected l-cysteine N-carboxyanhydride, respectively, in the presence of urea, at room temperature. The molecular weight of the PAA moiety was kept between M(n) = 2200 and 3000 g mol(-1). PMOx was polymerized by cationic ring-opening polymerization resulting in molecular weights of M(n) = 5000 and 10,000 g mol(-1), and PEG was a commercial product with M(n) = 5000 g mol(-1). Here, we investigate the self-assembly of the resulting amphiphilic block copolymers in water and the effect of the chemical structure of the block copolymers on the solution properties of self-assembled nanostructures. The PEG-block-poly(amino acid), PEG-b-PAA, and PMOx-block-poly(amino acid), PMOx-b-PAA, block copolymers have a narrow and monomodal molecular weight distribution (PDI < 1.3). Their self-assembly in water was studied by dynamic light scattering and fluorescence spectroscopy. In aqueous solution, the block copolymers associate into particles with hydrodynamic radii (R(H)) ranging in size from R(H) 70 to 130 nm, depending on the block copolymer architecture and the polymer molecular weight. Larger R(H) and critical association concentration values were obtained for copolymers containing poly(S-benzyloxycarbonyl-l-cysteine) compared to their poly(γ-benzyl-L-glutamate) analogue. FTIR investigations revealed that the poly(γ-benzyl-L-glutamate) block adopts a helical conformation, while the poly(S-benzyloxycarbonyl-L-cysteine) block exists as β-sheet.     

Base dynamics of nitroxide-labeled thymidine analogues incorporated into (dA-dT)n by DNA polymerase I from Escherichia coli

Nitroxide-labeled thymidine substrates (dL) for Escherichia coli DNA polymerase I (pol I) were used to synthesize spin-labeled alternating double-stranded copolymers with (dA-dT)n as a template. All dL substrates use an alkane or alkene tether substituted into the 5-position of the pyrimidine ring to link a five- or six-membered ring nitroxide to the pyrimidine base. The kinetics of dL incorporation show some tether dependence with respect to tether length and tether geometry. The electron spin resonance (ESR) spectra of (dA-dT,dL)n duplexes directly formed by polymerization with pol I are compared with the ESR spectra of (dA)n(dT,dL)n duplexes, which are obtained after annealing of nitroxide-labeled single strands with complementary unlabeled single strands. The ESR spectra indicate that nitroxide-labeled analogues with tethers short enough to let the nitroxide ring reside in the major groove are excellent reporter groups for monitoring hybridization. A small difference between the ESR line shapes of the alternating duplexes (dA-dT,dL)n and the homopolymer duplexes (dA)n(dT,dL)n containing the same dL is detectable, suggesting the presence of subtle differences in the base dynamics between both systems. Computer simulation of the ESR spectra of the (dA-dT,dL)n duplexes was successful with the same motional model reported earlier [Kao, S.-C., & Bobst, A.M. (1985) Biochemistry 24, 5465-5469]. The thymidine motion arising from tilting and torsion of base pairs and base twisting in (dA-dT)n is similar to that in (dA)n(dT)n and is of the order of 4 ns.     

Antisera to poly(A)-poly(U)-poly(I) contain antibody subpopulations specific for different aspects of the triple helix.

Rabbit antibodies to the triple-helical polynucleotide poly(A)-poly(U)-poly(I) were fractionated into three major antibody populations, each recognizing a different conformational feature of the triple-helical immunogen. Two distinct populations were purified from precipitates made with poly(A)-poly(U)-poly(U) and poly(A)-poly(I)-poly(I). The former reacted with double-stranded poly(A)-poly(U) or poly(I)-poly(C), and similar populations could be purified with either double-stranded form. The second population recognized the poly(A)-poly(I) region of the triple helix, and the third required all three strands for reactivity. These immunochemical studies suggest that the poly(A) and poly(U) have the same orientation in the triple-helicical poly(A)-poly(U)-poly(I) as in the double-helical poly(A)-poly(U), in which they have Watson-Crick base pairing.     

Immunochemical measurement of conformational heterogeneity of poly(inosinic acid).

Several pure poly(I) preparations differed in: (a) their complement fixation reactivity with anti-poly(I) antiserum; (b) their ability to bind to a solid-phase anti-poly(I) antibody-Sepharose column; (c) their ability to inactivate serum complement; and (d) their reactivity with purified antibodies to double-stranded RNA. In particular, poly(I) samples that could induce interferon production differed from non-inducer poly(I)s; the inducers reacted weakly with anti-poly(I) antiserum and were the only ones that reacted with antibodies to double-stranded RNA. One inducer poly(I) did not inactivate complement, and differed from non-inducer poly(I) in quantitative aspects of poly(I) . poly(C) formation with varying amounts of poly(C). An additional type of poly(I) preparation reacted poorly with anti-poly(I) antiserum, did not react with anti-double-stranded-RNA antibodies and failed to induce interferon production. The varying forms of poly(I) were not interconvertible by boiling and rapid chilling. These results indicate that several different stable structural forms of poly(I) may result from a standardized synthetic procedure.     

Interactions of a 5-nitroxide-labeled poly(uridylic acid) with poly(adenylic acid)

The physicochemical properties of a high-molecular-weight spin-labeled nucleic acid, (RUGT,U)_n, synthesized by enzymatic copolymerization, were evaluated by uv and ESR spectroscopy. It was shown earlier that spin labeling of nucleic acids by chemical modification to an extent which gives a nitroxide-to-nucleotide ratio greater than 0.002 can cause noticeable lattice perturbations (A. M. Bobst, A. Hakam, P. W. Langemeier, and S. Kouidou (1979), Arch. Biochem. Biophys. 187, 339–345). The presence of RUGT, a 5-nitroxide-labeled uridine residue, in a (U)_n lattice at a $\text{RUGT}\text{U}$ ratio of 0.01 is shown here not to affect the complexation with (A)_n, since the uv melting temperature (T₀^OD) of the 2 → 1 transition and the hypochromicity changes were the same for (RUGT,U)_n· (A)_n and (U)_n·(A)_n. ESR measurements indicated that the nitroxide radical reflects the transition accurately within the error limit, although a slight destabilization of the spinlabeled segment could not be excluded. Computer simulations showed conclusively that the spin melting temperature (T_m^sp) corresponds to the temperature at which half of the spin-labeled segments are no longer complexed, for the ESR spectrum at T_m^spcan be simulated with equal contributions from the line shapes of ESR spectra taken before and after the transition. Arrhenius plots obtained by using two different approaches for computing correlation times were qualitatively the same. Computer analysis also revealed that the formation of a (RUGT,U)_n·(A)_n complex can be described by a two-state model, in contrast to results obtained with chemically spin-labeled (U)_n. Thus, using (RUGT,U)_n over chemically spin-labeled (U)_n can offer distinct advantages.     

Proteins induced by the double-stranded interferon inducer poly(I).poly(C)

The possible pathways for realization of antiviral activity of interferon inducer poly (I).poly(C) have been studied. The stimulating effect of interferon inducer on the net protein synthesis in human M19 fibroblasts has been demonstrated. Compositions of the specific proteins induced by poly(I).poly(C) or interferon in human M19 fibroblasts and in monkey cells 4647 have been analyzed by electrophoresis technique. The data obtained suggest the existence of common gene products for interferon and ds-inducer. The ds-inducer requires the synthesis of lesser amounts of proteins for realization of its biological activity as compared with interferon.     

Effect of non-complementary nucleotides on the rate of helix formation: kinetics of formation of poly (I)-poly (C,I) and poly (I)-poly (C,U) complexes

Apparent second-order rate constants for complex formation between poly (I) and poly (C) and copolymers of C containing non-complementary I or U residues have been determined spectrophotometrically. The rate constants decrease as the concentration of either I or U in the C strands increases–the effect seems insensitive to the species of residue involved, when differences in the thermal stabilities of the poly (I) poly (C,I) and poly (I). poly (C,U) complexes are taken into account. These results suggest that low concentrations of relatively stable defects can alter the apparent kinetic “complexity” of polynucleotides as determined by hybridization methods (C₀t analysis).     

Spin labelling study of frozen solutions of porcine high density and low density lipoproteins: temperature dependence of the lipid dynamics

Frozen solutions of porcine lipoproteins, spin-labelled with steric acids, were studied as a function of temperature. High and low density lipoproteins and their subclasses were examined to obtain informatin on the relationship between the size, composition and dynamics of their lipid constituents. in low density lipoproteins (LDL) the temperature at which spin-labelled fatty acids responded to increased temperature depended on the position of the nitroxide moiety on the fatty acid chain. In LDL, e.s.r. spectra of 16-doxyl stearic acid I(1/14) with the nitroxide moiety buried depp in the phospholipid interior, responded to moderate increase of temperature even at ?50°C. In high densitylipoproteins (HDL), all spin-labelled fatty acids, I(m/n), remained in the frozen state (on the e.s.r. time scale) up to the melting point of the baffer. These differences in behaviour of the frozen lipoprotein solutions indicated that physicochemical properties of the surface constituents of the lipoprotein particle might be of a different nature in HLD and LDL.     

Interferon: a mediator of indoleamine 2,3-dioxygenase induction by lipopolysaccharide, poly(I) X poly(C), and pokeweed mitogen in mouse lung

When C3H/He mice were treated with lipopolysaccharide, poly(I) X poly(C), or pokeweed mitogen, the serum interferon titer increased almost instantaneously (100-2000 units/ml), and then the pulmonary indoleamine 2,3-dioxygenase was induced 50- to 140-fold. The peaks corresponding to interferon induction always preceded (approximately 24 h) those corresponding to dioxygenase induction. In C3H/HeJ (lipopolysaccharide-nonresponder) mice, however, lipopolysaccharide was totally inert in induction of both interferon and dioxygenase, although treatment with poly(I) X poly(C) and pokeweed mitogen led to a remarkable increase in the serum interferon titer and the enzyme activity. When lymphocytes of C3H/HeJ mice were inactivated by X irradiation and then reconstituted by the transfer of spleen cells from C3H/He mice, both enzyme and interferon from C3H/HeJ mice thus treated were induced almost normally after the lipopolysaccharide treatment. In addition, murine interferon alpha/beta, which was injected intravenously in C3H/He or C3H/HeJ mice, almost instantaneously and dose-dependently induced the pulmonary enzyme, and at a dose of 10(5) units per mouse the enzyme activity was enhanced 20- to 26-fold in these two strains of mice. These results suggest that interferon, which is generated by the interaction of lymphocytes with lipopolysaccharide, poly(I) X poly(C), or pokeweed mitogen, is a mediator of indoleamine 2,3-dioxygenase induction in the mouse lung by these agents.     

Nucleic acid interaction with VERO cells. A temperature barrier in the interaction pattern.

The interaction of VERO cell monolayers with spin (nitroxide)-(labeled polynucleotides (1(N)n) was examined by electron spin resonance (ESR) spectroscopy at various temperatures. Nitroxide labels covalently linked to (A)n, (dUfl)n, (U)n and (A)n . (U)n were used to monitor the interaction. The VERO cells were grown on small quartz plates with a cell viability of 95% or better and then used directly for the ESR studies. The ESR results indicated that the interaction between VERO cells and spin-labeled nucleic acids is temperature dependent. No temperature dependence was found when VERO cells were in contact with nitroxide radicals which were free in solution or covalently bound to Sepharose 4B. The temperature dependence established with nitroxide-labeled nucleic acids indicates that a temperature barrier must exist between 20 and 26 degrees C for the interaction between nucleic acids and VERO cells; namely, at 26 degrees C or above spin-labeled nucleic acids interact significantly with a VERO cell surface; whereas, at 20 degrees C the ESR signal reports no interaction. It is concluded that a temperature-dependent phase transition of membrane components or cell surface products active at 26 degrees C or above play a key role in the nucleic acid cell surface interaction process.     

Induction of antiviral activity in vivo and in vitro by human placenta ribonucleic acid treated with nitrous acid.

Induction of antiviral activity and interferon by human placenta ribonucleic acid deaminated with sodium nitrite (NO2-RNA) was studied in vitro and in vitro. (1) Viral multiplication in diploid cells from human kidney (HK cells) was depressed by pretreatment with NO2-RNA, but not by pre-treatment with the original placenta RNA. (2) NO2-RNA showed an interferon-inducing activity in rabbits and mice. (3) NO2-RNA sedimenting in 18 S and 28 S regions showed a higher antiviral activity than that sedimenting in 4 S region.