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).     

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.     

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.     

Hybridization of polymers of antibiotic C-nucleoside phosphates, poly(formycin phosphate) and poly(laurusin phosphate)

The ability of complex formation of poly-(formycin phosphate), poly(F), and poly(laurusin phosphate), poly(L), with the polymers of natural polynucleotides was examined mainly by mixing experiments in 0.1 M NaCl-0.05 M sodium cascodylate buffer (pH 7.0) at 2 degrees. Poly(F) formed complexes with poly(U) and poly(I) in the ratio of 1:1 and 1:2, respectively. Poly(L) formed complexes with poly(A) in 2:1 ration and poly(C) in 1:2 and 2:1 ratios in addition to a self-complex. Poly(F) and poly(L) also formed a 1:2 complex between them. Some of these complexes were assumed to contain novel types of base pairings using the 7-NH group. Thus it was concluded that poly(L) could form complexes with both, the oligomer of cycloadenylic acid (?cn-120 degrees) and polymers of natural nucleotides (?cn0degrees), showing flexibility of the torsion angle of the laurusin residue.     

Binding of novel 9-O–N-aryl/arylalkyl amino carbonyl methyl berberine analogs to poly(U)-poly(A)·poly(U) triplex and comparison to the duplex poly(A)-poly(U)

Interaction of the 9-O–N-aryl/arylalkyl amino carbonyl methyl substituted analogs of the anticancer isoquinoline alkaloid berberine with RNA triplex, poly(U)-poly(A)·poly(U) has been studied in comparison to the duplex poly(A)-poly(U), using multiple biophysical techniques. Spectrophotometric and spectrofluorimetric studies established the non-cooperative binding mode of all the analogs with both the duplex and the triplex. However, berberine exhibited cooperative binding with poly(A)-poly(U) and non-cooperative binding with poly(U)-poly(A)·poly(U). Analog BER1 showed the highest affinity to both the duplex and the triplex followed by BER2 and BER3. The overall binding affinity varied as BER1 > BER2 > BER3 > BER. The magnitude of the quantum efficiency values (Q > 1) revealed that energy was transferred from the bases of the triplex and the duplex to the analogs. Comparative ferrocyanide quenching and viscosity studies unambiguously established a stronger intercalative geometry of the analogs to both the triplex and the duplex in comparison to berberine. Circular dichroism studies revealed that the alkaloids perturbed the conformation of both RNA helices. The binding of all the alkaloids was found to be exothermic from isothermal titration studies. Binding of the analogs was highly entropy driven while that of berberine was enthalpy dominated. The results presented here reveal strong and specific binding of these new berberine analogs to the RNA triplex and duplex and highlight the remarkable influence of the 9-substitution on the interaction profile.     

Models of triple-stranded polynucleotides with optimised stereochemistry.

Detailed models are presented for the triple-stranded polynucleotide helices of poly (U)-poly (A)-poly (U) (two forms), poly (U)-poly d (A) -poly (U), poly d(C)-poly d(I)-poly d(C), poly d(T)-polyd(A)-poly d(T) and poly (I)-poly (A)-poly (I). The models were genrated using a computerized, linked-atom procedure which preserves standard bond lengths, bond anglesand sugar ring conformations, constrains the helices to have the pitches and symmetries observed in X-ray diffraction experiments, and optimises the non-bonded interatomic contacts including hydrogen bonds. The possible biological sigificance of such complexes is discussed.     

Interaction of metal ions with polynucleotides and related compounds. IX. Unwinding and rewinding of poly(A + U) and poly(I + C) by copper(II) ions

It is demonstrated that, poly(A + U) and poly(I + C) are both formed under low ionic strength conditions. Continuous variation studies indicate the formation of copper(II) complexes of poly A, poly C, and poly I, but not of poly U. Copper(II) in a 1:1 ratio to polynucleotide prevents the formation of poly(A + U) and brings about the dissociation of the poly (A + U) complex produced in the absence of the metal. Poly (I + C) is similarly dissociated by copper(II) ions. The addition of sufficient electrolyte reverses the copper(II) induced dissociation of poly(I + C). The effect of copper(II) on ordered synthetic polynucleotides is thus very similar to its effect on DNA.     

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.     

The binding of Mg(II) and Ni(II) to synthetic polynucleotides

Equilibria and kinetics of the interactions of Mg2+ and Ni2+ with poly(U), poly(C) and poly(I) have been investigated at 25 degrees C, an ionic strength of 0.1 M, and pH 7.0 or 6.0. Analogous studies involving poly(A) were reported earlier. All binding equilibria were studied by means of the (usually small) absorbance changes in the ultraviolet range. This technique yields apparent binding constants which are fairly large for the interaction of Ni2+ with poly(A) (K = 0.9 X 10(4) M-1) and poly(I) (K approximately equal to 2 X 10(4) M-1) but considerably lower for the corresponding Mg2+ systems, Mg2+-poly(A) (K = 2 X 10(3) M-1) and Mg2+-poly(I) (K = 280 M-1). Each of the two pyrimidine nucleotides binds both metal ions with about the same strength (K approximately equal to 65 M-1 for poly(U) and K near 600 M-1 for poly(C]. In the case of poly(C) the spectral changes deviate from those expected for a simple binding equilibrium. In addition, the binding of Ni2+ to the four polynucleotides was measured by using murexide as an indicator of the concentration of free Ni2+. The results obtained by this technique agree or are at least consistent with those derived from the ultraviolet spectra. Complications are encountered in the binding studies involving poly(I), particularly at higher metal ion concentrations, obviously due to the formation of aggregated poly(I) species. Kinetic studies of the binding processes were carried out by the temperature-jump relaxation technique. Measurable relaxation effects of time constants greater than 5 microseconds were observed only in the systems Ni2+-poly(A) and Ni2+-poly(I). Such not-too-fast reaction effects are expected for processes which include inner-sphere substitution steps at Mg2+ or Ni2+. The relaxation process in Ni2+-poly(I) is characterized by (at least) four time constants. Obviously, the complicated kinetics again include reactions of aggregated poly(I). The absence of detectable relaxation effects in all other systems (except Mg2+-poly(I), the kinetics of which was not investigated) indicates that inner-sphere coordination of the metal ions to specific sites of the polynucleotides (site binding) does not occur to a significant extent. Rather, the metal ions are bound in these systems mainly by electrostatic forces, forming a mobile cloud. The differences in binding strength which are nevertheless observed are attributed to differences in the conformation of the polynucleotides which result in different charge densities.     

Interferon induction by poly(inosinic acid).poly(cytidylic acid) segmented by spin-labels

Poly(inosinic acid).poly(cytidylic acid) [(I)n.(C)n] duplexes of which the (C)n strand was modified to various degrees chemically or enzymatically with nitroxide radicals (spin-labels) were evaluated for interferon-inducing activity. Upon annealing of the chemically modified (C)n, (1C,Cx)n (X = 1000 or 16), with (I)n, the interferon-inducing activity was similar to that of (I)n.(C)n in PRK cell cultures. However, to overcome hydrolysis of the spin-label linkage in (1C,Cx)n, and enzymatic approach was taken to synthesize (1S(4)U,Cx)n copolymers with x = 100, 38, 16, and 8. The (1s(4)U,Cx)n copolymers were chemically stable, and upon annealing with (I)n the correlation time of the nitroxide moiety in (I)n. (1s(4)U,Cx)n was determined. A comparison of this correlation time with that measured for (RUGT,U100)n.(A)n, which contains the nitroxide moiety in position 5 of the U moiety, suggests that the 1s(4)U residue is in a nonintrahelical conformation and partitions the duplex into double-helical segments of varying size. The interferon-inducing activity of (I)n. (1s(4)U,Cx)n was evaluated in primary rabbit kidney, human skin fibroblast (strain VGS), and mouse L-929 cell cultures as well as in rabbits. The 1s(4)U residue did not cause a significant change in the interferon induction as compared to (I)n.(C)n in most systems tested unless x less than 16. These findings indicate that double-helical segments of approximately 16 base pairs partitioned by nonintrahelical 1s(4)U residues suffice to trigger the interferon response in all systems studied.     

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.     

Polynucleotides XLVII. Synthesis and properties of poly(2-methylthio- and 2-ethylthioadenylic acid). Formation of non-Watson-Crick type complexes.

Poly(2-methyl- and 2-ethylthioadenylic acid) were prepared by polymerization of corresponding diphosphates with Escherichia coli polynucleotide phosphorylase. These polynucleotides have relatively large hypochromicity of 30-35%. Acid titration of these polymers showed abrupt transition at pH 5.34-5.4, which may indicate that the introduction of alkylthio group at 2-position of adenine bases reduced their basicity. Thermal melting of these polymers showed no clear transition points at neutral pH, but in acidic media they have Tm values of 57 and 56 degrees C, somewhat lower than that of poly(A). Upon complex formation with poly(U), these poly(A) analogs showed only one poly(rs2A) . poly(U) type double-strand complexes, similar to that found in the case of poly(m2A) . poly(U).     

Antitumour activity of endotoxin, concanavalin A and poly I: C and their ability to elicit tumour necrosis factor, cytostatic factors, and interferon in vivo

Summary Concanavalin A, endotoxin, poly I : C, and tumour necrosis serum (TNS) were compared for antitumour activity against Meth A sarcoma transplanted in syngeneic BALB/c mice and their capacity to induce tumour necrosis factor (TNF), heat-stable cytostatic factors, and heat-labile interferon in the blood of normal and Corynebacterium parvum-pretreated mice. All the agents induced hyperemia and inhibition of mitosis at 4 h, and by 24 h many tumours had a dark necrotic centre. Subsequent tumour growth was inhibited and in some of the treated mice tumours regressed completely. Poly A : U and normal mouse serum did not induce regression and their effects were less marked in all other respects, suggesting that these events may be linked. The necrotizing effects of concanavalin A and poly I : C are unlikely to be mediated by TNF, because neither agent could mimic endotoxin in eliciting RNase-resistant necrotizing and regressing activity in the serum of mice pretreated with C. parvum. Poly I : C did not induce strong cytostatic activity in the sera of C. parvum-treated mice, and for this and other reasons these factors are unlikely to be responsible for the observed effects. Concanavalin A, endotoxin, and poly I : C induced high levels of serum interferon but purified interferon had only weak antitumour activity in the Meth A system, suggesting that interferon may not be the mediator.From these and other data it is concluded that there is no clear relationship between the capacity of the agents to induce tumour necrosis and their capacity to elicit TNF, cytostatic factors, and interferon.     

Induction by synthetic polyribonucleotide poly(I) of differentiation of cultured mouse myeloid leukemic cells.

The effects of some synthetic polyribonucleotides on induction of differentiation of mouse myeloid leukemic M1 cells were examined. Poly(I) was found to be a potent inducer; on treatment with 100--200 microgram/ml of poly(I) for 2--4 days, M1 cells differentiated into cells resembling macrophages and granulocytes and developed phagocytosis and locomotive activities, Fc receptors and lysozyme activity. Poly(C) was less effective than poly(I) for induction of phagocytic activity, while the other single-stranded RNAs, poly(U) and poly(A), had no effect. Double-stranded RNAs, such as poly(I) . poly(C) and poly(A) . poly(U), were cytotoxic to M1 cells, and differentiation of the cells could not be detected even at the highest tolerable concentrations of these double-stranded RNAs.     

Sepharose-bound poly (I)-poly (C): interaction with cells and interferon production.

Poly(I).poly(C) covalently coupled to a matrix by one point fixation through its 3′ terminal stimulated both antiviral activity and interferon production in primary rabbit kidney (PRK) cells. This effect could not be accounted for by free polynucleotide released from the matrix into the medium. Penetration of the polynucleotide into the cells does not appear to be necessary for interferon production. A limited amount of matrix-bound poly(I).poly(C) was associated with the cells. Since it was sensitive to extraneous ribonuclease treatment, this poly(I).poly(C) was believed to be localized at the cell surface. Preliminary findings suggest that the binding of the polynucleotide to the cell is not directly proportional to the amount of interferon induced.     

Protective effect of poly I:poly C from gentamicin nephrotoxicity in guinea pigs

Poly(inosinic) and poly(cytidylic) acids (Poly I:Poly C) have been used to induce the production of endogenous interferon or release preformed interferon in mammals. Interferon increases the resistance of the cells. Sixty guinea pigs were used to investigate whether Poly I:Poly C gave protection from gentamicin nephrotoxicity. The animals were divided into six equal groups. Group 1 were controls; group 2 received gentamicin intramuscularly; group 3 received gentamicin and 12 h later frusemide; group 4 received gentamicin and 12 h later 1-deamino-8-D-argine vasopressin (DDAVP) intramuscularly; group 5 received subcutaneously Poly I:Poly C; group 6 received Poly I:Poly C and 24 h later gentamicin. Frusemide in group 3 potentiated gentamicin nephrotoxicity while DDAVP in group 4 ameliorated gentamicin nephrotoxicity. Poly I:Poly C itself had no toxic effect on renal tissue, while Poly I:Poly C followed 24 h later by gentamicin indicated a protective effect from the gentamicin nephrotoxicity as the functional and histological investigations indicated.     

Dissociation of double-stranded poly(I) . poly(C) by cis-diammine-dichloro-Pt(II).

The covalent binding of cis-Pt(NH3)2Cl2 on the double stranded poly(I) . poly(C) induced an irreversible dissociation of the two strands. This dissociation was evidenced mainly by poly(I)-Agarose affinity chromatography which allowed to recover free strands of cis-Pt(NH3)2Cl2-poly(I) from a cis-Pt(NH3)2Cl2-poly(I) . poly(C) complex, by density equilibrium centrifugation where free poly(C) could be isolated, and by acid titrations of the metal-poly(I) . poly(C) complexes. The separation of the two strands of the polyribonucleotide upon cis-Pt(NH3)2Cl2 fixation was shown not to exceed 90--95%. A dissociation curve of the polynucleotide double helix as a function of the amount of bound cis-Pt(NH3)2Cl2 was determined and was shown to be of a characteristic cooperative effect. The fixation of the paltinum compound to poly(I) . poly(C) seemed also to be cooperative.     

Differential susceptibilities of DNA polymerases-alpha and -beta to polyanions.

The effects of various polyanions including synthetic polynucleotides on DNApolymerases-alpha and -beta from blastulae of the sea urchin Hemicentrotus pulcherrimus and HeLa cells were studied. Only DNA polymerase-alpha was inhibited by polyanions, such as polyvinyl sufate, dextran sulfate, heparin, poly(G), poly(I), poly(U) and poly(ADP-Rib). Of the various polynucleotides tested, poly(G) and poly(I) were the strongest inhibitors. Kinetic studies showed that the Ki value for poly(G) was 0.3 microgram/ml and that poly(G) had 20-fold higher affinity than activated DNA for the template-primer site of DNA polymerase-alpha. Poly(U) and poly(ADP-Rib) were also inhibitory, but they were one hundredth as inhibitory as poly(G) or poly(I). Poly(A), poly(C), poly(A).poly(U) AND POLY(I).poly(C) were not inhibitory to DNA polymerase-alpha. In contrast, DNA olymerase-beta was not affected at all by these polyanions under the same conditions.     

A premelting conformational transition in poly(dA)-Poly(dT) coupled to daunomycin binding

Circular dichroism and UV absorbance spectroscopy were used to monitor and characterize a premelting conformational transition of poly(dA)-poly(dT) from one helical form to another. The transition was found to be broad, with a midpoint of tm = 29.9 degrees C and delta HVH = +19.9 kcal mol-1. The transition renders poly(dA)-poly(dT) more susceptible to digestion by DNase I and facilitates binding of the intercalator daunomycin. Dimethyl sulfoxide was found to perturb poly(dA)-poly(dT) structure in a manner similar to temperature. These combined results suggest that disruption of bound water might be linked to the observed transition. A thermodynamic analysis of daunomycin binding to poly(dA)-poly(dT) shows that antibiotic binding is coupled to the polynucleotide conformational transition. Daunomycin binding renders poly(dA)-poly(dT) more susceptible to DNase I digestion at low binding ratios, in contrast to the normal behavior of intercalators, indicating that antibiotic binding alters the conformation of the polynucleotide. The unusual thermodynamic profiles previously observed for the binding of many antibiotics to poly(dA)-poly(dT) can be explained by our results as arising from the coupling of ligand binding to the polynucleotide conformational transition. Our data further suggest a physical basis for the temperature dependence of DNA bending.