Template-directed oligomerization catalyzed by a polynucleotide analog.

A pyrophosphate-linked analog of polycytidylic acid has been synthesized and shown to catalyze the oligomerization of the complementary monomer 2'-deoxyguanosine 3',5'-bisphosphoimidazolide. Analogs of polynucleotides are of interest in studies of the origins of life as possible precursors of the first RNA molecules. These results demonstrate that such molecules are capable of serving as templates for further synthesis.

Action of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver.

The effects of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver were investigated. The enzyme was isolated from the nuclear fraction essentially according to the method of Baril et al.; it was characterized as the alpha polymerase on the basis of its response to synthetic templates and its inhibition with N-ethylmaleimide. Although polycytidylic acid had no effect on the DNA polymerase alpha either as a template or as an inhibitor, partially thiolated polycytidylic acid (MPC) was found to be a potent inhibitor, its activity being directly related to its extent of thiolation (percentage of 5-mercaptocytidylate units in the polymer). In comparison, the DNA polymerase beta which was purified from normal rat liver nuclear fraction, was much less sensitive to inhibition by MPC. Analysis of the inhibition of the alpha polymerase by the method of Lineweaver and Burk showed that the inhibitory action of MPC was competitively reversible with the DNA template, but the binding of the 7.2%-thiolated MPC to the enzyme was much stronger than that of the template (Ki/Km less than 0.03). Polyuridylic acid as such showed some inhibitory activity which increased on partial thiolation, but the 8.4%-thiolated polyuridylic acid was less active than the 7.2% MPC. When MPC was annealed with polyinosinic acid, it lost 80% of its inhibitory activity in the double-stranded configuration. However, 1 to 2%-thiolated DNA isolates were significantly more potent inhibitors than were comparable (1.2%-thiolated) MPC and showed competitive reversibility with the unmodified (but "activated") DNA template. These results indicate that the inhibitory activities of partially thiolated polynucleotides depend not only on the percentage of 5-mercapto groups but also on the configuration, base composition, and other specific structural properties.

Antiviral effects of single-stranded polynucleotide inhibitors of the influenza virion-associated transcriptase against influenza virus infection of hamsters and ferrets.

Administration of a single-stranded polynucleotide copolymer containing 9% cytidine residues and 91% 4-thiouridine residues [poly(C,S4U10)], a known potent inhibitor of the virion transcriptase of influenza viruses, suppressed the amount of virus recoverable from the nasal washes of influenza virus-infected hamsters and ferrets. The incidence of sneezing and nasal discharge in infected ferrets was also reduced. In hamsters, poly(C,S4U10) was more effective than amantadine-HCl or Virazole. Polyinosinic acid in combination with poly-5-hydroxy cytidylic acid also had anti-influenza effects. Poly(C,S4U10) annealed to polyadenylic acid was not effective, nor was the double-stranded polymer (polyinosinic acid) . (polycytidylic acid) even when complexed with carboxymethylcellulose and polylysine. No toxic effects of poly(C,S4U10) were apparent in the treated hamsters and ferrets, and high doses (greater than or equal to 2.86 g/kg) administered intraperitoneally to mice produced no adverse effects.

Polynucleotides containing 5-mercapto-substituted pyrimidines: inhibition of viral DNA polymerases and the biological implication.

Partially thiolated polycytidylic acids MPC I-III, containing 1.7%, 3.5% and 8.6% 5-mercaptocytidylate units, respectively) inhibited the DNA polymerase of Friend leukemia virus (FLV) in the endogenic reaction as well as in the presence of poly(A)-(dT)14 or poly[d(a-T)] templates; the inhibitory activities were directly related to the percent of thiolation. Various partially thiolated RNA and DNA isolates from Ehrlich ascites cells (containing one 5-mercaptopyrimidine nucleotide/50-100 nucleotide units) also inhibited the DNA polymerases of FLV in the endogenic reaction, and also in the presence of the synthetic templates. The thiolated DNA was the most active, but the thiolated tRNA also showed substantial inhibitory effects, while the thiolated ribosomal RNA was less effective. In a bacterial DNA polymerase (E. coli-K12, using denatured DNA as template), MPC I-III showed no activity. By contrast, MPC III and several partially thiolated nucleic acid isolates significantly inhibited a regenerating rat liver DNA polymerase (I) system; among those tested, the thiolated DNA from Ehrlich ascites cells showed the highest activity. Kinetic analysis of the inhibitory action of this thiolated DNA in the rat liver enzyme system, using as template the corresponding unmodified DNA, demonstrated that the thiolated DNA acts as a competitive inhibitor of the template, with a Ki/Km ratio of 0.5.

Fluorescence decay and quantum yield characteristics of acridine orange and proflavine bound to DNA.

Fluorescence properties (quantum yield, decay curve, lifetime and polarization) of acridine orange and proflavine bound to DNA were examined as a function of nucleotide to dye (P/D) ratio. First, mean fluorescence lifetimes were determined by the phase-shift measurements. The lifetime and quantum yield of acridine orange increased in a parallel fashion with increasing P/D ratio. There was no parallel relation between the lifetime and quantum yield for proflavine; the lifetime showed a minimum around P/D equals 10...

Poly-2'-deoxy-2'-fluoro-cytidylic acid: enzymatic synthesis, spectroscopic characterization and interaction with poly-inosinic acid.

The polymerization of 2'deoxy-2'-fluoro-cytidine-diphosphate (dCflDP) by polynucleotide phosphorylase is barely detectable in the presence of Mg++ under usual experimental conditions for polymerization of nucleoside diphosphates. High concentrations of enzyme have to be used to accomplish the synthesis. Mn++ is a better activator than Mg++ for the reaction. cCflDP inhibits the polymerization of CDP and has a Km=8.8X10-3M, six times higher than CDP.- The polymer, poly (dCfl), ressembles in many respects poly(C), but not poly(dC): the acid selfstructure forms at similar pK's; interaction with poly(I) yields a 1:1 complex the CD spectrum of which is similar to that of poly(I).poly(C). Finally, the Tm's of poly(I).poly(dCfl) are comparable to those of poly(I).poly(C).

Poly 2'-O-ethylcytidylate, an inhibitor and poor template for AMV reverse transcriptase.

Poly(2'-O-ethylcytidylate) is a poor template-primer for purified avian myeloblastosis virus reverse transcriptase; the relative activities of the template-primers poly(C)-oligo(dG), poly(Cm)-oligo(dG) and poly(Ce)-oligo(dG) are 23:16:1. A mixture of poly(Ce) and poly(dI) is inactive as template-primer, in agreement with the observed inability of these to form a helical complex. By contrast the inactivity of poly(Ce)-poly(I) is shown to be due to the influence of the 2'-O-ethyl residue. Poly(Ce) inhibits poly(A)-oligo(dT)-directed polymerase activity, with Ki = 3 muM, but marked inhibition with poly(A)-poly(dT) occurs only at low concentrations of the latter. Poly(Ce) did not inhibit template-primer activity of poly(C)-poly(dI) and poly(dC)-poly(dI). Qualitative physico-chemical studies show only partial complex formation between oligo(dG) and poly(C) and its 2'-O-alkyl analogues. This is discussed in relation to the widespread use of poly(C)-oligo(dG) as the template-primer for reverse transcriptase.

Preparation and properties of poly 2'-O-ethylcytidylic acid.

Poly 2'0-ethylcytidylic acid (poly (Ce)) was prepared by polymerization of 2'-0-ethylcytidine-5'-pyrophosphate with Escherichia coli polynucleotide phosphorylase in the presence of Mn++, and its properties compared with those of poly (rC), poly (Cm) and poly (dC). The neutral form of pOLY (Ce) exhibits properties similar to those of poly (rC) and poly (Cm). It also forms an acid twin-stranded helix with a transition pH of 5.9 in 0.1 M NaCl. The neutral form readily forms a double-stranded helical complex with poly (rI). Relative to poly (Cm), replacement of the 2'-0-methyl by 2-0-ethyl leads to increased enhancement of the thermal stabilities of both the acid helical form of poly (Ce) and its complex with poly (rI).

Role of purine N-3 in the biologic activities of poly(A) and poly(I).

Poly(c3A) (poly 3-deazaadenylic acid) and poly(c3I) (poly 3-deazainosinic acid) differ in biological reactivity from their parent compounds poly(A) and poly(I) and from their 7-deaza counterparts poly(c7A) and poly(c7I). Three parameters of biological reactivity were evaluated : (1 degree) interferon induction, (2 degrees) anti-complement activity, (3 degrees) reverse transcriptase inhibition. Unlike poly(A)-poly(U), poly(I)-poly(C) and poly(I)-poly(br5C), the mixtures of poly(c3A) + POLY(U), poly(c3I) + poly(C), and poly(c3I) + poly(br5C) failed to elicit an interferon response in "super-induced" primary rabbit kidney cells; Poly(I) and its analogs poly(c3I) and poly(c7I) inhibited hemolytic complement activity, whereas poly(A) and its analogs poly(c3A) and poly(c7A) failed to do so. Both poly(I) and poly(c7I), but not poly(c3I), lost their anti-complement potency when annealed to either poly(C) or poly(A)-poly(U). Similarly, poly(I) and poly(c7I), but not poly(c3I), suppressed the interferon inducing ability of poly(A)-poly(U), suggesting that both poly(I) and poly(c7I), but not poly(c3I), added to poly(A)-poly(U) to form a triple-helical structure. Poly(I), poly(C7I) and poly(c7A)exerted a distinct inhibitory effect on turine leukemia virus, while under the same conditions poly(c3I) and poly(c3A) showed little, if any, inhibitory effect.