Polynucleotides. LVII. Synthesis and properties of poly (2''-chloro-2''-deoxyinosinic acid).

Poly (2'-chloro-2'-deoxyinosinic acid) [poly(Icl)] was synthesized from Icl 5'-DP by polymerization with polynucleotide phosphorylase. UV absorption properties of poly(Icl) are very similar to those of poly(I). Poly(Icl) adopted a multi-stranded ordered form in the presence of 0.95M Na ion. The Tm value of this form was 36 degrees, which resembles that of poly(I) quadruple-stranded form at high salt. CD spectra also suggested presence of these two forms. Upon mixing with poly(C), poly-(Icl) forms a double-stranded 1 : 1 complex, which had very similar Tm-log[Na+] relationship to that of poly(I) . poly(C). Thus it was concluded that the chlorine substitution at 2'-position of the polynucleotide had the similar effect to OH on physical properties of polynucleotides.     

Polynucleotides. LVI. Synthesis and properties of poly(2-deoxy-2''-fluoroinosinic acid).

Poly (2'-deoxy-2'-fluoroinosinic acid) [ poly(If)] was synthesized by polymerization of 2'-deoxy-2'-fluoroinosine 5'-diphosphate catalyzed by Escherichia coli polynucleotide phosphorylase. Although the UV absorption properties of poly(If) closely resembled those of poly(I), thermal melting curves at Na+ concentrations of 0.15M and 0.75M suggested two ordered structures for poly(If) neutral form. CD psectra taken at 0.15M Na+ concentration showed rather larger amplitudes in both a peak at 273 nm and a trough at 246 nm, suggesting rather strong vertical stacking of bases. When complexed with poly(C), poly(If) forms a double-stranded complex, poly(If).poly(C) which has Tm's higher by 10-20 degrees than those of poly(If).poly(C) measured under the same conditions. The CD spectrum of this complex resembled that of poly(I).poly(C). The effect of the fluorine atom at the 2'-position on thermal stability of polynucleotides is discussed.     

Poly(2-methylthio-7-deazainosinic acid)--hydrophobic stabilization of polynucleotide secondary structure by the 2-methylthio group.

Poly(2-methylthio-7-deazainosinic acid) [poly(ms2c7I)] was enzymatically synthesized by polymerization of 2-methylthio-7-deazainosine 5'-diphosphate with polynucleotide phosphorylase from Micrococcus luteus in high yield. The homopolymer shows much higher thermal stability than its parent polynucleotides poly(7-deazainosinic acid) [poly(c7I)] and poly(I). Its sigmoidal melting curve and pronounced hypochromicity imply a rigid, ordered structure. Poly(ms2c7I), like poly(2-methylthio-inosinic acid) [poly(ms2I)], does not form a complex with poly(C) because of the bulky 2-methylthio substituent. On the other hand, two poly(ms2c7I) strands form very rigid triple strands with poly(A). Different from poly(I) and poly(c7I) the homopolymer poly(ms2c7I) is very stable against cleavage by nuclease S1 and ribonuclease T2 as expected from its rigid secondary structure.     

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.     

Fluorescent 2-aza-epsilon-adenosine derivatives of polyadenylic acid, polyuridylic acid, and polyinosinic acid.

A new fluorescent analog of adenosine, 1,N⁶-etheno-2-aza-adenosine, has been incorporated into polynucleotides by polynucleotide phosphorylase polymerization of 1,N⁶-etheno-2-aza-adenosine-5′-diphosphate and adenosine-5′-diphosphate, uridine-5′-diphosphate, or inosine-5′-diphosphate. These new oligonucletides possess high fluorescence when excited at 358 nm and emit at 495 nm. The ratio of the fluorescent and nonfluorescent portions of the copolymer can be controlled by the initial composition of the 2-aza-ε-adenosine-diphosphate and the corresponding nucleoside diphosphate. Fluorescent copolymers with a ratio varying from 1.6 to 35 have thus been synthesized. The physicochemical study of copolymers containing less than 10% of the 1,N⁶-etheno-2-aza-adenosine moiety showed that they are similar to poly(A), poly(U), or poly(I). Therefore, fluorescence and polarization study of the 1,N⁶-etheno-2-aza-adenosine residues that have been incorporated into the copolymer provides a sensitive indicator for the structure of the copolymer. Potentially these new copolymers may provide unique roles in probing the structure of poly(C) and poly(A) in cellular mRNA.     

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).     

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.     

Adenylate kinase of plants. Properties of adenylate kinase of pea leaves]

The properties of adenylate kinase in 2 ADP in equilibrium ATP + AMP reaction have been studied. The dependence of the enzyme activity on medium pH, protein concentration, substrates, Mg++ ions, AMP, adenine and adenosine has been also investigated. pH optimum is found to be 8.5 for forward reaction and 8-9--for the reverse one. The Michaelis constants are as follows: for ADP--1.17-10(-4) M, for ATP--3.33-10(-4) M at 24 degrees C, in 50 mM tris-HCl pH 7.6. The optimal ratio, Mg++ ions/substrates (ADP, ATP + AMP), is 1:2. The chelates of adenine nucleotides with Mg++ ions are proved to be "true" reaction substrates. Unlike adenine and adenosine, the product of AMP reaction inhibits adenylate kinase activity. It is concluded that the properties of adenylate kinase in plants are similar to those of animals and humans (moikinase).     

Poly(2-fluoroadenylic acid). The role of basicity in the stabilization of complementary helices.

The polymerization of 2-fluoroadenosine 5'-diphosphate by polynucleotide phosphorylase to give high molecular weight poly(2-fluoroadenylic acid), poly(fl2A), is described. Both the single-stranded and double-stranded (acid) forms of poly(fl2A) exhibit strikingly similar ultraviolet and circular dichroism spectra to those of poly(A), and the enzymatic polymerization rates and thermal hyperchromicities of the two polymers are also very similar. However, the pKa of poly(fl2A) for protonation at N-1 is 2.9 compared to 5.9 for poly(A) under similar conditions. Poly(fl2A) forms a triple-stranded helix with poly(U) which has ultraviolet and cd spectra very reminiscent of poly(A) . 2 poly(U), but no conditions could be found which permitted the formation of a double helix. In the Escherichia coli ribosome system poly(fl2A) codes for the synthesis of polylysine, as does poly(A), although the rate and extent of incorporation were less in the former case. The role of basicity of adenine N-1 in these interactions is discussed.     

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).     

The viral mimic, polyinosinic:polycytidylic acid, induces fever in rats via an interleukin-1-dependent mechanism

Polyinosinic:polycytidylic acid (poly I:C) is a synthetic double-stranded RNA that is used experimentally to model viral infections in vivo. Previous studies investigating the inflammatory properties of this agent in rodents demonstrated that it is a potent pyrogen. However, the mechanisms underlying this response have not been fully elucidated. In the current study, we examined the effects of peripheral administration of poly I:C on body temperature and cytokine production. Male rats were implanted with biotelemetry devices and randomly assigned to one of the following three groups: poly I:C + saline, poly I:C + interleukin-1 receptor antagonist (IL-1ra), or saline + saline. Maximal fever of 1.6 degrees C above baseline was observed 3 h after an intraperitoneal injection of poly I:C (750 microg/kg). Pretreatment with IL-1ra diminished this response by >50% (maximum body temperature = 0.6 degrees C above baseline). Plasma IL-6 concentration increased fivefold 2 h post-poly I:C compared with saline-injected rats; levels returned to baseline 4 h postinjection. Pretreatment with IL-1ra prevented this rise in IL-6. Plasma tumor necrosis factor (TNF)-alpha was also increased more than fourfold 2 h postinjection but remained unaffected by IL-1ra treatment. IL-1beta and cyclooxygenase-2 mRNA were significantly upregulated in the hypothalamus of poly I:C-treated animals. Finally, poly I:C decreased food intake by 30%, but this response was not altered by pretreatment with IL-1ra. These results suggest that poly I:C induces fever, but not anorexia, through an IL-1 and prostaglandin-dependent mechanism.     

Polynucleotides. XXII. Synthesis and properties of poly 7-deazainosinic acid

Poly 7-deazainosinic acid has been prepared by the deamination and phosphorylation of tubercidin and the nucleoside diphosphate was polymerised using polynucleotide phosphorylase. The polymer has similar physical properties to poly(I), but has a low thermal stability in the double-stranded complex with poly(C). Poly(7-deaza I), in contrast, forms a more stable triple-stranded complex with poly(A) than 2 poly(I). poly(A), presumably due to the higher pK value.     

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.     

Polynucleotides. XL. Synthesis and properties of poly 2''-azido-2''-deoxyadenylic acid.

Poly 2'-azido-2'-deoxyadenylic acid (Poly Az) was synthesized from 2'-azido-2'-deoxyadenosine diphosphate by polynucleotide phosphorylase. Poly (Az) has U.V. absorption properties similar to poly (A) and hypochromicity of 40% at 0.1 M Na+ and neutrality. CD curve also resembled to that of poly (A), but has smaller ellipticity. Titration of poly (Az) with HCl gave a transition at pH 5.5, but exact structure of the acid-form complex was not elucidated. Upon mixing with poly (U), poly (Az) forms a 1:1 and 1:2 complexes having Tm's somewhat higher than that of poly (A)- poly (U) complex in the same condition.     

Accumulation of 2'',5''-oligoadenylates in encephalomyocarditis virus-infected mice.

Levels of 2',5'-oligoadenylates (2-5A) in various tissues of murine encephalomyocarditis virus (EMCV)-infected mice were determined and compared with those found in pathogen-free mice and in mice treated with the interferon inducer poly(I).poly(C). In control, pathogen-free mice, liver, spleen, brain, and kidney tissues possessed levels of 2-5A below 1 pmol/g of tissue, demonstrating that 2-5A was not a major component of uninfected mouse tissue. All control tissues had low basal levels (0.3 to 2.0 pmol/h per g) of 2-5A synthetase, the enzyme responsible for 2-5A production. After mice were injected intravenously with the interferon inducer poly(I).poly(C), circulating interferon, 2-5A synthetase, and 2-5A were elevated with increasing doses of double-stranded RNA. The greatest response to poly(I).poly(C) occurred in the kidney, in which enzyme levels increased 5-fold and 2-5A levels increased 24-fold to 15 pmol/g. Mice that were infected with EMCV also possessed elevated levels of 2-5A and 2-5A synthetase in the four tissues examined, although the relative distribution differed from that observed with poly(I).poly(C), indicating that the interferon inducer affects the concentration and location of intracellular 2-5A. Brain, spleen, and kidney tissues from EMCV-infected mice contained seven- to eightfold more 2-5A than control tissues did. The nanomolar levels of 2-5A in the tissues of EMCV-infected mice provide evidence that 2-5A may play a role in the antiviral response in an intact animal. In both poly(I).poly(C)- and EMCV-treated mice, the levels of 2-5A recovered from the tissues were not directly proportional to the amount of 2-5A synthetase present. These results indicate that factors other than the level of 2-5A synthetase controlled the accumulation of 2-5A in tissues.     

Mg2+ and Ni2+ ion effect on stability and structure of triple poly I.poly A.poly I helix

The effects of Mg2+ and Ni2+ ions on the absorption spectra of IMP, single-stranded poly I and three-stranded A2I in solutions with 0.1 M Na+ (pH 7) have been studied. In contrast to Mg2+ ions, the Ni2+ ions affect the absorption spectra of these polynucleotides and IMP. The concentration dependences of the intensity at the extrema in the differential UV spectra suggest that in the region of high Ni2+ concentrations ionic complexes with poly I and A2I are formed, which are characterized by the association constants K'I = 2000 M(-1) and K'A2I = 550 M(-1), respectively. The shape of the DUV spectra prompts the conclusion that these complexes are formed due to the inner-sphere interaction of Ni2+ ions with N7 of poly I and A2I presumably due to the outer-sphere Ni2+-O6 interaction. The formation of the complexes leads to destruction of A2I triplexes. The dependences of the melting temperature (T(m)) of A2I on Mg2+ and Ni2+ concentrations have been measured. The thermal stability is observed to increase at the ionic contents up to 0.01 M Mg2+ and only to 2x10(-4) M Ni2+. At higher contents of Ni2+ ions, T(m) lowers and the cooperativity of A2I melting decreases continuously. In all the cases the melting process is the A2I-->A+I+I (3-->1) transition. According to the "ligand" theory, these effects are generated by the energy-advantageous Ni2+ binding to single-stranded poly I (K'A2I < K'I) and by the greater number of binding sites which appears during the 3-->1 transition and is entropy-advantageous.     

Phase equilibrium in poly(rA).poly(rU) complexes with Cd2+ and Mg2+ ions, studied by ultraviolet, infrared, and vibrational circular dichroism spectroscopy

Ultraviolet (UV) and infrared (IR) absorption and vibrational circular dichroism (VCD) spectroscopy were used to study conformational transitions in the double-stranded poly(rA). poly(rU) and its components-single-stranded poly(rA) and poly(rU) in buffer solution (pH 6.5) with 0.1M Na+ and different Mg2+ and Cd2+ (10(-6) to 10(-2) M) concentrations. Transitions were induced by elevated temperature that changed from 10 up to 96 degrees C. IR absorption and VCD spectra in the base-stretching region were obtained for duplex, triplex, and single-stranded forms of poly(rA) . poly(rU) at [Mg2+],[Cd2+]/[P] = 0.3. For single-stranded polynucleotides, the kind of conformational transition (ordering --> disordering --> compaction, aggregation) is conditioned by the dominating type of Me2+-polymer complex that in turn depends on the ion concentration range. The phase diagram obtained for poly(rA) . poly(rU) has a triple point ([Cd2+] approximately 10(-4)M) at which the helix-coil (2 --> 1) transition is replaced with a disproportion transition 2AU --> A2U + poly(rA) (2 --> 3) and the subsequent destruction of the triple helix (3 --> 1). The 2 --> 1 transitions occur in the narrow temperature interval of 2 degrees -5 degrees . Unlike 2 --> 1 and 3 --> 1 melting, the disproportion 2 --> 3 transition is a slightly cooperative one and observed over a wide temperature range. At [Me2+] approximately 10(-3) M, the temperature interval of A2U stability is not less than 20 degrees C. In the case of Cd2+, it increases with the rise of ion concentration due to the decrease of T(m) (2-->3). The T(m) (3-->1) value is practically unchanged up to [Cd2+] approximately 10(-3)M. Differences between diagrams for Mg(2+) and Cd2+ result from the various kinds of ion binding to poly(rA).poly-(rU) and poly(rA).