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.     

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.     

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. LII1 Synthesis and properties of poly(2′-deoxy-2′-fluoroadenylic acid)

2′-Deoxy-2′-fluoroadenosine was chemically transformed to its 5′-diphosphate and polymerized with polynucleotide phosphorylase to give poly(2′-deoxy-2′-fluoroadenylic acid) [poly(Af)]. Polymerization proceeded smoothly as in the case of poly(A) and the yield of the polymerization was 55%. The UV absorption spectra of poly(Af) closely resembled those of poly(A) and the hypochromicity was 32% at pH 7.0. The CD profile at 25° and neutrality showed similar pattern to that of other poly(2′-deoxy-2′-halogenoadenylic acids) with somewhat larger [θ] values both in the positive and negative maxima. Acid titration of poly(Af) showed a transition point at pH 5.2 and the Tm of the acid form was 37° which was significantly lower than that of poly(A), but similar to that of poly(2′-azido-2′-deoxyadenylic acid). Poly(Af) formed 1:1 and 1:2 complexes with poly-(U) having Tm of 49° and 62° at 0.04M and 0.15M Na⁺ concentration, respectively. Poly(Af) also formed a 1:2 complex with poly(I) and its Tm was 36° at 0.05M Na⁺ concentration. These data showed that poly(Af) has rather similar properties to those of poly(A), but not to poly(dA).     

Studies on polyribonucleotides. Synthesis of a polyinosinic: 6-thioinosinic acid copolymer

The enzymatic synthesis of a high molecular weight (s_20,w ～10) copolymer of inosinic and 6-thioinosinic acids [poly(I:s⁶I)] has been achieved. Poly (I:s⁶I) forms a double stranded complex with poly C which has a Tm significantly lower than a poly I · ply C complex of equivalent molecular weight.     

Interactions of poly-N6-methyladenylic acid and N6-substituted-9-methyladenines with poly-5-bromouridylic acid: a novel base-pairing

The interaction of poly-N6-methyladenylic acid (poly(m6A) with poly-5-bromouridylic acid (poly(BU) was studied by the mixing curve method. A.1 m6A: 2 BU stoichiometry was clearly indicated over a wide range of ionic strengths at neutral pH, while the binding of poly(m6A) to poly(U) is known to occur with 1 m6A:1 U. Digestion by nuclease S1 confirmed this stoichiometry, indicating the absence of single strands in a 1:2 mixture. Heating profile analysis and hydroxyapatite column chromatography provided further confirmation of this finding. To determine whether 1:2 stoichiometry holds in a monomer-polymer system, the interaction of N6-methyl-9-methyladenine (m6m9A), a corresponding monomer of poly(m6A), with poly(BU) was investigated. Equilibrium dialysis experiments showed the stoichiometry of the interaction to be 1 m6A:2 BU. Thus, we would describe some structural studies of the above complexes using c.d. and i.r. spectroscopy. Poly (m6A).2poly(BU) and m6m9A.2poly(BU) are helical and analogous to each other in structure, and the bases in the complexes are all bound by hydrogen-bonding. N6-(delta 2-isopentenyl)- and N6-allyl-9-methyladenine were also found to form complexes with poly(BU), giving similar c.d. spectra with that of m6m9A.2poly(BU). The melting experiments indicated the Tms to be substantially decreased, compared to the parent unmodified complexes, even though the Tm dependence of the polymer complex on salt concentration conforms to the typical triple strand. In the following, the biological significance of this novel pairing will be discussed.     

Polyribonucleotides containing thiopurines: Synthesis and properties

The synthesis of poly(1-methyl-6-thioinosinic acid) and a comparison of its properties with those of poly(6-thioinosinic acid) and poly(6-methylthiopurinylic acid) are reported. In contrast to 6-thioinosine 5′-diphosphate, 1-methyl-6-thioinosine 5′-diphosphate was found to be a substrate for polynucleotide phosphorylase-catalyzed homopolyribonucleotide synthesis. Poly(1-methyl-6-thioinosinic acid) appears to form a single stranded helical array with a highly cooperative melting transition (Tm = 12°C) and a very large bathochromic shift (12 nm) in the absorption maximum upon melting.     

Nonionic nucleic acid analogues. Synthesis and characterization of dideoxyribonucleoside methylphosphonates.

A series of dideoxyribonucleoside methylphosphonate analogues, dNpN and dNpNp, which contain a nonionic 3'--5' methylphosphonyl internucleoside linkage were prepared. The two diastereoisomers, designated isomers 1 and 2, of each dimer differ in configuration of the methylphosphonate group and were separated by column chromatography. The diastereoisomers of each dimer have different conformations in solution as shown by ultraviolet hypochromicity data and their circular dichroism spectra. For example, dApA isomer 1 is more highly stacked than isomer 2, although both isomers are less stacked than the dinucleoside monophosphate, dApA. The circular dichroism spectrum of isomer 1 is very similar to that of dApA, while the CD spectrum of isomer 2 shows a loss of molecular ellipticity, [theta], at 270 nm and a greatly diminished [theta] at 250 nm. These results suggest that the stacked bases of dApA isomer 1 tend to orient in an oblique manner, while those in isomer 2 tend to orient in a parallel manner. This interpretation is verified by the 1H NMR study of these dimers (L. S. Kan, D. M. Cheng, P. S. Miller, J. Yano, and P. O. P. Ts'o, unpublished experiments). Both diastereoisomers of dAaA form 2U:1A and 2T:1A complexes with poly(U) and poly(dT), respectively. The higher Tm (Tm of poly(U)--isomer 1, 15.4 degrees C; Tm of poly(U)--isomer 2, 19.8 degrees C; Tm of poly(dT)--isomer 1, 18.7 degrees C; Tm of poly(dT)--isomer 2, 18.4 degrees C) values of these complexes vs. those of the corresponding dApA--polynucleotide complexes (Tm of poly(U)--dApA, 7.0 degrees C; Tm of poly(dT)--DApA, 9.2 degrees C) result from decreased charge repulsion between the nonionic dimer backbone and the negatively charged polymer backbone. The difference in conformations between dApA isomer 1 and dApA isomer 2 is reflected in the Tm of the isomer 1-poly(U) complex which is 4.4 degrees C lower than that of the isomer 2-poly(U) complex. Since these nonionic oligonucleotide analogues are taken up by cells in culture, they show promise as molecular probes for the function and structure of nucleic acids inside living cells.     

The interaction of 2,6,8-triaminopurine with poly(uridylic acid).

Equilibrium dialysis measurements have shown that poly(uridylic acid) binds 2,6,8-triaminopurine in a strongly cooperative manner to form a stable 2 : 1 complex at pH 7.8, 0.15 M Na+. The thermal dissociation of the complex has been characterized by ultraviolet absorbance versus temperature profiles. From the variation of Tm with the concentration of uncomplexed triaminopurine at this temperature, the partial molar enthalpy and entropy of formation of the complex have been calculated as --87 (+/- 2) kJ/mol of triaminopurine and --236 (+/- 7) J/mol of triaminopurine per K, respectively. In terms of Tm, the complex is approximately 4 degrees C more stable than the corresponding 2 : 1 complex of poly(U) with 2-aminoadenine. This stabilization is attributed to the existence of an additional hydrogen-bonding interaction, in the poly(U)-triaminopurine complex, between the 8-amino group of 2,6,8-triaminopurine and O(2) of the uracil moiety which is base paired with it in Hoogsteen fashion.     

Effect of DEAE-dextran upon the interaction of polynucleotides in poly (l+c) double complex]

It has been shown that the formation of poly(I+C) double complex is accompanied by appearance of the 244 nm CD band which is absent from the spectrum of the initial components. The amplitude of this CD band is maximum upon equimolar ratio of components. When one mixes the complementary polynucleotides bound to DEAE-dextran (D-d) double comples is not formed. CD spectrum of poly (I+C) double complex is changed considerably upon addition of D-d: CD increases when P/N ratio is 10:1, decreases at P/N 1:1 and comes back to the initial spectrum at P/N 1:5. Thermal dissociation of poly(I+C) when the anionic component was in surplus was similar to poly(I+C) alone (Tm equals 67 degrees) when the polydextran was in excess; the thermal dissociation was lower (Tm equals 43 degrees) than that of poly (I+C). It is discussed the possible mechanism of the D-d and poly (I+C) interaction.     

Polynucleotides. XLVI. 1 Synthesis and properties of poly (2''-amino-2''-deoxyadenylic acid).

Poly (2'-amino-2'-deoxyadenylic acid) [poly (Aa)] was prepared from chemically synthesized 2'-amino-2'-deoxy-ADP by the catalysis of polynucleotide phosphorylase. Poly (Aa) showed a similar UV absorption spectra to poly (A), but quite different CD spectra at pH 7.0 and 5.7. At the former pH it showed a single negative Cotton band and at the latter a curve with a large splitting of bands. Acid titration of poly (Aa) suggested protonated form below pH 7.0. Temperature absorption profiles and their dependency on sodium ion concentration suggested an ordered structure for poly (Aa) which is stabilized by stacking of bases and intrastrand interaction between 2'-amino and internucleotidic phosphate groups. Poly (Aa) forms a 1:2 complex with poly (U) at neutrality and its Tm was 45 degrees in the presence of 0.15M sodium ion.     

Microcalorimetric investigation of DNA, poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] melting in the presence of water soluble (meso tetra (4 N oxyethylpyridyl) porphyrin) and its Zn complex

Thermodynamic parameters of melting process (DeltaHm, Tm, DeltaTm) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C)).poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased Tm of satellite fraction by 24 degrees C, but ZnTOEpyp(4), on the contrary, predominantly bound with AT-rich sites and increased DNA main stage Tm by 18 degrees C, and Tm of poly(dA)poly(dT) increased by 40 degrees C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes--strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode--strong binding--took place. The weak binding is characterized with shifting of Tm by some grades, and for the strong binding Tm shifts by approximately 30-40 degrees C. Invariability of DeltaHm of DNA and poly(dA)poly(dT), and sharp increase of Tm in the range of r from 0.08 to 0.25 for thymus DNA and 0.01-0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H2O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width DeltaTm caused by increase of added ZnTOEpyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which DeltaT approximately TGC-TAT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193-205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.     

Complex formation between ribosomal protein S1, oligo-and polynucleotides: chain length dependence and base specificity.

In order to examine the nature of the complex formation between the ribosomal protein S1 and nucleic acids three methods were used: Inhibition of the reaction of n-ethyl[2.3 14C]-maleimide with S1 by the addition of oligonucleotides; adsorption of the complexes to nitrocellulose filters; and equilibrium dialysis. The complex formation is Mg2+ dependent at low salt concentrations and becomes Mg2+ independent at an ionic strength greater than 90 mM. Oligouridylates of increasing chain length reach an optimal KA of 3-3-10(7) M-1 at a chain length of n=13-14. Protein S1 contains one binding site for long chain oligouridylates, such as U12, and the standard-free-energy change on binding caused by one Pu increment is 0.41 kcal/mol, when n varies between five and fourteen. Complex formation is insensitive to the capacity of the homopolynucleotide bases to form hydrogen bonds. Homopolynuceotides, however, showing a Tm less than 250 in the buffer system used show an increased affinity for S1 compared to poly(A) and poly(C) (Tm greater than 40 degrees). The data are discussed with respect to the proposed binding of protein S1 to the 3-terminal end of the 16S RNA.     

Physical and coding properties of poly(5-methoxyuridylic) acid.

The synthesis of poly(mo5U) requires a high concentration (2.7 mg/ml) of polynucleotide phosphorylase as well as a long reaction time (48 h). The resulting polynucleotide has a chain length of approximately 100 nucleotides. It shows no indication of a stable secondary structure. When poly(mo5U) is mixed with poly(A), a triple-stranded complex poly(A) . 2poly(mo5U) is formed. This complex has a melting temperature of 68.5 +/- 0.5 degrees C at 150 mMNa+ and exhibits a hysteresis loop between melting and reformation of the complex having a delta Tm of 11.5 degrees C. Poly-5-methoxyuridylic acid stimulates the binding of Phe-tRNA to 70-S ribosomes but is inactive in directing poly(Phe) synthesis.     

A basic isozyme of yeast glyceraldehyde-3-phosphate dehydrogenase with nucleic acid helix-destabilizing activity

A nucleic acid helix-destabilizing protein has been purified from Saccharomyces cerevisiae using affinity chromatographic techniques. Crude protein extracts at low ionic strength (approx. 0.05 M) were applied sequentially to tandem columns of native DNA-cellulose, aminophenyl-phosphoryl-UMP-agarose, poly(I . C)-agarose, poly(U)-cellulose and denatured DNA-cellulose. The 2 M NaCl eluant of the poly(U)-cellulose column was dialyzed to low ionic strength and recycled through native DNA-cellulose, poly(I . C)-agarose and poly(U)-cellulose. Purified helix-destabilizing protein eluted from the poly(U)-cellulose between 0.1 and 0.5 M NaCl. On the basis of enzymatic activity, immunological cross-reactivity, mobility on SDS gels, amino acid analysis and preliminary peptide mapping experiments, this material was identified as an isozymic fraction of glyceraldehyde-3-phosphate dehydrogenase. The major crystallizable isozyme of this enzyme from yeast is, however, considerably more acidic than the helix-destabilizing protein, and displays significantly lower helix-destabilizing activity. Stoichiometric levels of the isolated protein at low (approx. 0.01) ionic strength depress the Tm of poly(A-U) and poly [d(A-T)] by as much as 28 and 22 degrees C, respectively. Longer double helices, poly(A . U) and Clostridium perfringens DNA are also denatured by the helix-destabilizing protein, but at relatively slow rates. The binding of this protein to [3H]-poly(U) on nitrocellulose filters in [Na+]-dependent, with a 50% reduction at 0.09 M NaCl. Based on its effect on the circular dichroism spectrum of poly(A), the protein was shown to distort the conformation of the polynucleotide chain. An analogous protein from mammalian cells, P8, was also shown to depress poly(A-U) Tm.     

Virus-Associated Nucleases: Location and Properties of Deoxyribonucleases and Ribonucleases in Purified Frog Virus 3

At least three nuclease activities are associated with purified frog virus 3. These activities are endodeoxyribonuclease (pH 7.5, double-stranded [DS] and single-stranded [SS] deoxyribonucleic acid [DNA]); endodeoxyribonuclease (pH 5.0, DS and SS DNA); endoribonuclease (DS and SS ribonucleic acid [RNA], pH 7.5). These activities are not adsorbed to the surface of the virion but are within the viral capsid and require detergent disruption of virions to unmask enzyme activity. Only one activity, deoxyribonuclease (pH 5.0, SS and DS DNA) appears to be core-associated after detergent disruption of virions. The ribonuclease degrades poliovirus replicative-form RNA, reovirus native RNA, and poly(I) poly(C) to a product with a sedimentation coefficient of about 6S. Qbeta 6S DS RNA and 4S transfer RNA are not degraded. The ribonuclease appears to be a late function of the virus and is elicited in a soluble form as well as a virus-associated form.     

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.     

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.     

Altered fractionation profiles of higher plant poly(A)-containing RNA by selective formation of a complex with a hydrophobic impurity

Synthetic polyadenylate (poly(A)) which originally sedimented at 6–7 S in a sucrose density gradient did so at 15–19 S in the presence of phenol-extracted nucleic acids from higher plant tissues. After centrifugation, the input poly(A) was found to be insensitive to RNase T2 (EC 3.129.1), suggesting a newly-formed complex with a hydrophobic substance contaminating the nucleic acid preparation. The artifically-formed poly(A)-impurity complex migrated more slowly than unbound poly(A) during gel electrophoresis. The complex was retained rather effectively by an oligodeoxythymidylate (oligo-(dT))-cellulose column and a polyuridylate (poly(U))-glassfiber filter. However, a nitrocellulose membrane filter retained the poly(A)-complex to various extents depending on plant materials, suggesting different varieties of the poly(A)-binding impurity in the higher plant kingdom. Fractionation of mRNA by these ordinarily used procedures is suggested to be variously altered by complex formation with this hydrophobic impurity.