Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs.

The stability and structure of RNA duplexes with consecutive A.C, C.A, C.C, G.G, U.C, C.U, and U.U mismatches were studied by UV melting, CD, and NMR. The results are compared to previous results for GA and AA internal loops [SantaLucia, J., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819; Peritz, A., Kierzek, R., & Turner, D.H. (1991) Biochemistry 30, 6428-6436)]. The observed order for stability increments of internal loop formation at pH 7 is AG = GA approximately UU greater than GG greater than or equal to CA greater than or equal to AA = CU = UC greater than or equal to CC greater than or equal to AC. The results suggest two classes for internal loops with consecutive mismatches: (1) loops that stabilize duplexes and have strong hydrogen bonding and (2) loops that destabilize duplexes and may not have strong hydrogen bonding. Surprisingly, rCGCUUGCG forms a very stable duplex at pH 7 in 1 M NaCl with a TM of 44.8 degrees C at 1 x 10(-4) M and a delta G degrees 37 of -7.2 kcal/mol. NOE studies of the imino protons indicate hydrogen bonding within the U.U mismatches in a wobble-type structure. Resonances corresponding to the hydrogen-bonded uridines are located at 11.3 and 10.4 ppm. At neutral pH, rCGCCCGCG is one of the least stable duplexes with a TM of 33.2 degrees C and delta G degrees 37 of -5.1 kcal/mol. Upon lowering the pH to 5.5, however, the TM increases by 12 degrees C, and delta G degrees 37 becomes more favorable by 2.5 kcal/mol. The pH dependence of rCGCCCGCG may be due to protonation of the internal loop C's, since no changes in thermodynamic parameters are observed for rCGCUUGCG between pH 7 and 5.5. Furthermore, two broad imino proton resonances are observed at 10.85 and 10.05 ppm for rCGCCCGCG at pH 5.3, but not at pH 6.5. This is also consistent with C.C+ base pairs forming at pH 5.5. rCGCCAGCG and rGGCACGCC have a small pH dependence, with TM increases of 5 and 3 degrees C, respectively, upon lowering the pH from 7 to 5.5. rCGCCUGCG and rCGCUCGCG also show little pH dependence, with TM increases of 0.8 and 1.4 degrees C, respectively, upon lowering the pH to 5.5.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential hydration of homopurine sequences relative to alternating purine/pyrimidine sequences.

The minor groove ligand distamycin A has been used to probe the relative hydration of the minor groove of eight synthetic polynucleotides of known sequence and composition. A combination of densimetric, calorimetric, and temperature-dependent spectroscopic techniques have been used to obtain complete thermodynamic profiles (delta Gzero, delta Hzero, delta Szero, and delta Vzero) for the association of distamycin A to all polymer duplexes. In 10 mM phosphate buffer, pH 7, binding of the drug to each of the polymeric duplexes resulted in characteristic negative changes in both the volume and enthalpy. Although the binding constants were found to be identical for pairs of isomer polynucleotides having identical compositions but different sequences, the values of delta Hzero, delta Szero, and delta Vzero of each such pair were remarkably different. The entropy changes were found to roughly parallel the volume changes; no such trend was seen between delta Hzero and delta Vzero. The data support the hypothesis that the volume changes observed for these systems reflect the coulombic-hydration contribution to the entropy. The heteropolymer duplexes generated much larger exothermic contributions, less favorable entropies and larger volume contractions than did the corresponding homopolymer duplexes of identical composition, and strongly suggest that polynucleotides with homopurine sequences are more hydrated than polynucleotides with alternating purine/pyrimidine sequences. In addition, it was found that duplexes containing guanine sharply reduced the affinity for the drug, also lowering the exothermicity but raising the entropy. This may be explained by the presence of an amino group in the minor groove that prevents hydrogen bonding. Substitution of the guanine with inosine reversed this trend in the thermodynamic properties. Furthermore, substitution of poly(dA) for poly(rA) in a duplex produced a similar reduction in the affinity, while raising the exothermic contribution and greatly reducing the favorable entropy effect in agreement with an apparent increase in the hydration state.     

Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine

A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed. Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy. A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes. The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair. On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair. It is concluded that s2Um has higher selectivity toward A over G than unmodified U.     

Thermodynamics of DNA branching.

Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is + 1.1 (+/- 0.4) kcal mol-1 (at 18 degrees C, 10 mM-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 degrees C is +27.1 (+/- 1.3) kcal mol-1, while the entropy is +89 (+/- 30) cal K-1 mol-1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent delta Cp is also observed for the formation of one mole of junction, +0.97 (+/-0.05) kcal deg-1 mol-1.     

Sterol substrate specificity of acyl coenzyme A:cholesterol acyltransferase from the corn earworm, Heliothis zea

The enzymatic activity and sterol substrate specificity of acyl coenzyme A:cholesterol acyltransferase (ACAT) were measured in microsomes of cells from Heliothis zea. Under standard assay conditions, the specific enzymatic activity of ACAT was highest in the intestine followed by the fat body and ovary (380.7, 30.7, 8.3 pmol/min per mg, respectively). The structure of the exogenous sterol used in the ACAT assay affected its rate of esterification. The relative rates of esterification of analogs of cholesterol with various modifications of the side chain were: 24-H greater than 24 alpha-CH3 greater than delta 22 greater than delta 24 greater than 24 alpha-C2H5 greater than 24 beta-CH3, delta 22-24 beta-CH3 and delta 22-24 alpha-C2H5. The number and position of double bonds in the B-ring of the sterol nucleus greatly affected the rate of esterification of sterols by ACAT. The average relative rates of esterification of sterols with differences in their B-rings were: delta 7 much greater than delta 8 greater than delta 0 greater than delta 5 greater than delta 5.7. The presence of a 9,14-cyclopropane group and/or methyl groups at the C-4 and 14 positions prevented significant esterification of such sterols. The formation of cholesteryl and lathosteryl esters was partially inhibited in microsomes from the intestine, fat body, and ovary by the addition of the ACAT inhibitor, 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]prop anamide (Sandoz Compound 58-035).(ABSTRACT TRUNCATED AT 250 WORDS)     

Watson-Crick base-pairing properties of bicyclo-DNA.

A series of sequences of the DNA analog bicyclo-DNA, 6-12 nucleotides in length and containing all four natural nucleobases, were prepared and their Watson-Crick pairing properties with complementary RNA and DNA, as well as in its own series, were analyzed by UV-melting curves and CD-spectroscopy. The results can be summarized as follows: bicyclo-DNA forms stable Watson-Crick duplexes with complementary RNA and DNA, the duplexes with RNA generally being more stable than those with DNA. Pyrimidine-rich bicyclo-DNA sequences form duplexes of equal or slightly increased stability with DNA or RNA, whereas purine-rich sequences show decreased affinity to complementary DNA and RNA when compared with wild-type (DNA-DNA, DNA-RNA) duplexes. In its own system, bicyclo-DNA prefers antiparallel strand alignment and strongly discriminates for base mismatches. Duplexes are always inferior in stability compared with the natural ones. A detailed analysis of the thermodynamic properties was performed with the sequence 5'-GGATGGGAG-3'x 5'-CTCCCATCC-3' in both backbone systems. Comparison of the pairing enthalpy and entropy terms shows an enthalpic advantage for DNA association (delta deltaH = -18 kcal x (mol)-1)) and an entropic advantage for bicyclo-DNA association (delta deltaS = 49 cal x K(-1) x mol(-1), leading to a delta deltaG 25 degrees C of -3.4 kcal x mol(-1) in favor of the natural duplex. The salt dependence of Tm for this sequence is more pronounced in the case of bicyclo-DNA due to increased counter ion screening from the solvent. Furthermore bicyclo-DNA sequences are more stable towards snake venom phosphodiesterase by a factor of 10-20, and show increased stability in fetal calf serum by a factor of 8 compared with DNA.     

Synthesis and properties of oligonucleotides containing 2''-deoxynebularine and 2''-deoxyxanthosine.

The preparation of synthetic oligonucleotides containing 2'-deoxynebularine (dN) and 2'-deoxyxanthosine (dX) is described. The thermal stabilities of duplexes containing dX, dN, and 2'-deoxyinosine (dI) base-paired with the four natural bases have been measured. Xanthine base pairs have stabilities at pH 5.5 that are similar to those of dI-containing duplexes at neutral pH. When xanthine is paired with adenine or cytosine an unusual stabilization of the duplex structure is observed at acid pH. Incorporation of base mispairs opposite template xanthine sites were measured using Drosophila DNA polymerase alpha. The relative nucleoside incorporation rates are in the order: T greater than C much greater than A approximately equal to G. These rates do not correlate with relative thermodynamic stabilities of base mispairs with xanthine obtained from Tm measurements: T greater than G greater than A approximately equal to C. We suggest that DNA polymerase misinsertion rates are greatest when the base mispair can be formed in accordance with Watson-Crick as opposed to other base pairing geometries even though other geometries, e.g. wobble, may result in a more stable final DNA product.     

Electron transfer in DNA duplexes containing 2-methyl-1,4-naphthoquinone

2-Methyl-1,4-naphthoquinone (menadione, MQ) was linked to synthetic oligonucleotides and exposed to near-UV light to generate base radical cations in DNA. This model system of electron transfer induced alkali-labile breaks at GG doublets, similar to anthraquinone and metallointercalators systems. In sharp contrast to other systems, the photolysis of MQ–DNA duplexes gave interstrand cross-links and alkali-labile breaks at bases on the complementary strand opposite the MQ moiety. For sequences with an internal MQ, the formation of cross-links with A and C opposite the MQ moiety was 2- to 3-fold greater than that with G and T. The yield of cross-links was more than 10-fold greater than that of breaks opposite MQ, which in turn was more than 2-fold greater than breaks at GG doublets. The yield of damage at GG doublets greatly increased for a sequence with a terminal MQ. The distribution of base damage was measured by enzymatic digestion and HPLC analysis (dAdo > dThd > dGuo > dCyd). The formation of novel products in MQ–DNA duplexes was attributed to the ability of excited MQ to generate the radical cations of all four DNA bases; thus, this photochemical reaction provides an ideal model system to study the effects of ionizing radiation and one-electron oxidants.     

Low enantioselectivities of human deoxycytidine kinase and human deoxyguanosine kinase with respect to 2'-deoxyadenosine, 2'-deoxyguanosine and their analogs

The antiviral activity of L-nucleoside analogs depends in part on the enantioselectivity of nucleoside kinases which catalyse their monophosphorylation. The substrate properties of human recombinant deoxycytidine kinase (dCK) and human recombinant deoxyguanosine kinase (dGK) with respect to L-adenosine and L-guanosine analogs, in the presence of saturating amounts of ATP and relatively high concentrations of substrates, demonstrated a marked lack of enantioselectivity of both these enzymes. Human dCK catalysed the phosphorylation of D- and L-enantiomers of beta-dA, beta-araA, and beta-dG with enantioselectivities favoring the unnatural enantiomer for the adenosine derivatives and the natural enantiomer for 2'-deoxyguanosine. No other tested L-adenosine or L-guanosine analog was a substrate of dCK. Similarly, D- and L-enantiomers of beta-dA, beta-araA, and beta-dG were substrates of human dGK but with different enantioselectivities compared to dCK, especially concerning beta-dA. The present results indicate that human dCK and dGK have similar properties including substrate properties, relaxed enantioselectivities, and possibly catalytic cycles.     

Calculation of the stability of mini-duplexes of DNA in an electrolyte solution using a molecular mechanics method in approximating a statistical model of the environment

Approximation of the statistical model of environment (SME) to estimate the energy of the macromolecule in electrolyte solution has been developed and used for calculating the conformational energy of nucleic acids by means of the molecular mechanics method. Calculation of base pairs opening delta Hcalop enthalpies and enthalpies of DNA miniduplexes dissociation delta Hcaldis were performed for 10 types of diduplexes. The approximation SME enable to perform calculations of the absolute base-dependent values of delta Hcalop which coincide in the range of 1 kcal/mol with the experimental base-dependent values of the helix-coil transition enthalpies. Values of dissociation enthalpies delta Hcalop greater than delta Hcaldis for all miniduplexes, the difference of delta Hcalop--delta Hcaldis determine the base-dependent energy of the helix-coil boundary. The values of activation barriers for the strands dissociation delta H d not equal to congruent to 8 kcal/mol and association delta H not equal to as congruent to 4 kcal/mol were obtained for GG/CC and AA/TT duplexes. It is concluded that the approximation SME enables to increase substantially the accuracy of the calculations of the macromolecule conformational rearrangement enthalpies in the electrolytes solution.     

Isolation of sealed plasma membrane vesicles from Phytophthora megasperma f. sp. glycinea. I. Characterization of proton pumping and ATPase activity

Sealed vesicles were isolated from a plant pathogenic fungus Phytophthora megasperma f. sp. glycinea using a modification of a method previously developed for plant plasma membrane vesicle isolation. Vanadate-sensitive, proton pumping microsomal membrane vesicles were resolved on a linear sucrose density gradient and found to comigrate with a vanadate-sensitive ATPase. Both the proton pumping and ATPase activity of these vesicles had a pH optimum of 6.5 and demonstrated similar properties with respect to substrate specificity and inhibitor sensitivity. These properties were in agreement with previously published data on the Phytophthora plasma membrane ATPase. In contrast with previous reports there was no K+ stimulation of the plasma membrane ATPase and the Km for Mg:ATP (1:1 concentration ratio) was higher (2.5 mM). A comparison of anion (potassium salts) effects upon delta pH and delta psi formation in sealed Phytophthora plasma membrane vesicles revealed a correspondence between the relative ability of anions to stimulate proton transport and to reduce delta psi. The relative order for this effect was KCl greater than KBr much greater than KMes, KNO3, KClO3, K2SO4. This study presents a method for the isolation of sealed vesicles from Phytophthora hyphae. It also provides basic information on the plasma membrane H+-ATPase and its associated proton pumping activity.     

Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide.

The thermal stabilities of RNA:DNA hybrids are substantially greater than those of DNA:DNA duplexes in aqueous electrolyte solutions containing high concentrations of formamide. Association rates to form DNA:DNA duplexes and DNA:RNA hybrids have been measured in these solvents. There is a temperature range in which DNA:DNA rates are negligible and RNA:DNA rates close to optimal.     

NADH formation by Na+-coupled reversed electron transfer in Klebsiella pneumoniae

Citrate is fermented by Klebsiella pneumoniae to 2 acetate, 0.5 formate and 1.2 CO2. The formation of less than 1 formate and greater than 1 CO2 per citrate can be accounted for by the oxidation of formate to CO2 in order to provide reducing equivalents for the assimilation of citrate into cell carbon. A membrane-bound electron transport chain is apparently involved in NADH synthesis by these cells. The electrons from formate oxidation to CO2 are used to reduce ubiquinone to ubiquinol by membrane-bound formate dehydrogenase and ubiquinol further delivers its electrons to NAD+, if this endergonic reaction is powered by delta mu Na+. The endogenous NADH level of K. pneumoniae cells thus increased in the presence of formate in response to a delta pNa+ greater than -100 mV. NADH formation was completely abolished in the presence of oxygen or after addition of hydroxyquinoline-N-oxide, a specific inhibitor of the Na(+)-translocating NADH:ubiquinone oxidoreductase. The increase of endogenous NADH was dependent on the delta pNa+ applied to the cells. Inverted membrane vesicles of K. pneumoniae catalysed the reduction of NAD+ to NADH with formate as electron donor after application of delta mu Na+ of about 120 mV consisting of delta pNa+ of 60 mV and delta psi of the same magnitude. Neither the delta pNa+ nor the delta psi of this size alone was sufficient to drive the endergonic reaction. Strictly anaerobic conditions were required for NADH formation and hydroxyquinoline-N-oxide completely inactivated the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of site-specific substitution of 5-fluorouridine on the stabilities of duplex DNA and RNA.

The effects of 5-fluorouridine (FUrd) and 5-fluorodeoxyuridine (FdUrd) substitution on the stabilities of duplex RNA and DNA have been studied to determine how FUrd substitution in nucleic acids may alter the efficiency of biochemical processes that require complementary base pairing for molecular recognition. The parent sequence, 5'-GCGAAUUCGC, contains two non-equivalent uridines. Eight oligonucleotides (four RNA and four DNA) were prepared with either zero, one or two Urd substituted by FUrd. The stability of each self-complementary duplex was determined by measuring the absorbance at 260 nm as a function of temperature. Tm values were calculated from the first derivative of the absorbance versus temperature profiles and values for delta H0 and delta S0 were calculated from the concentration dependence of the Tm. Individual absorbance versus temperature curves were also analyzed by a parametric approach to calculate thermodynamic parameters for the duplex to single-stranded transition. Analysis of the thermodynamic parameters for each oligonucleotide revealed that FUrd substitution had sequence-dependent effects in both A-form RNA and B-form DNA duplexes. Conservation of helix geometry in FUrd-substituted duplexes was determined by CD spectroscopy. FUrd substitution at a single site in RNA stabilized the duplex (delta delta G37 = 0.8 kcal/mol), largely due to more favorable stacking interactions. FdUrd substitution at a single site in DNA destabilized the duplex (delta delta G37 = 0.3 kcal/mol) as a consequence of less favorable stacking interactions. All duplexes melt via single cooperative transitions.     

Base pairing involving deoxyinosine: implications for probe design.

The thermal stability of oligodeoxyribonucleotide duplexes containing deoxyinosine (I) residues matched with each of the four normal DNA bases were determined by optical melting techniques. The duplexes containing at least one I were obtained by mixing equimolar amounts of an oligonucleotide of sequence dCA3XA3G with one of sequence dCT3YT3G where X and Y were A, C, G, T, or I. Comparison of optical melting curves yielded relative stabilities for the I-containing standard base pairs in an otherwise identical base-pair sequence. I:C pairs were found to be less stable than A:T pairs in these duplexes. Large neighboring-base effects upon stability were observed. For example, when (X,Y) = (I,A), the duplex is eight-fold more stable than when (X,Y) = (A,I). Independent of sequence effects the order of stabilities is: I:C greater than I:A greater than I:T congruent to I:G. This order differs from that of deoxyguanosine which pairs less strongly with dA; otherwise each deoxyinosine base pair is less stable than its deoxyguanosine counterpart in the same sequence environment. Implications of these results for design of DNA oligonucleotide probes are discussed.     

Cloning and functional expression of a cDNA encoding a metabolic acyl-CoA delta 9-desaturase of the cabbage looper moth, Trichoplusia ni.

Acyl-CoA delta 9-desaturases play essential roles in fatty acid metabolism and the regulation of cell membrane fluidity. In this research, a cDNA sequence was obtained from Trichoplusia ni adult fat body mRNA by using RT-PCR with degenerate primers based on other characterized delta 9-desaturase sequences. The remainder of the sequence was amplified using 3'- and 5'-RACE. A 1439 bp cDNA reconstructed from three overlapping PCR products contains an ORF encoding a 353-amino acids (aa) protein that shows clear homology (greater than 50% aa identity and greater than 65% aa similarity to characterized insect and vertebrate desaturases). The ORF of this cDNA was subcloned into an expression vector, which relieved the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient yeast strain following genetic transformation. The newly characterized desaturase from T. ni produced fatty acids delta 9-16 and delta 9-18 in a 1:6 ratio, compared to a 5:1 ratio, respectively, with the yeast delta 9 desaturase. A Northern blot hybridization and a RT-PCR experiment showed that temporal and tissue-specific patterns of expression of the corresponding mRNA are distinct from those of the delta 11-desaturase mRNA present in the pheromone glands of adult females. Based on its homology to other desaturases, the widespread distribution of its corresponding mRNA in various tissues, and its functional assay, we conclude that this cDNA encodes the apoprotein corresponding to the desaturase component of the metabolic delta 9-desaturase complex of T. ni.     

Studies on nucleic acid interactions. I. Stabilities of mini-duplexes (dG2A4XA4G2-dC2T4YT4C2) and self-complementary d(GGGAAXYTTCCC) containing deoxyinosine and other mismatched bases.

The thermal stability of DNA duplexes containing deoxyinosine in a pairing position in turn with each of the four major deoxynucleotides has been investigated by measuring ultraviolet-absorbance at different temperatures. d(G2A4 X A4G2) and d(C2T4YT4C2) were prepared by the solid-phase phosphotriester method. When X is deoxyinosine, the Tm values of the duplexes are in the order Y = dC greater than dA greater than dG greater than dT greater than dU. The Tm of other duplexes containing dG, dA and dT at X were also measured. Self-complementary duplexes d(GGGAAINTTCCC) showed the same order of stability with N being dC, dA, dG and dT. Thermal stabilities of duplexes containing dG instead of dI were compared with other matched and mismatched duplexes. The Tm values of sequence isomers containing purine-pyrimidine combinations were compared. Self-complementary duplexes containing G-C and A-T in the central positions showed Tm values ca. 10 degrees higher than those containing C-G and T-A in the same positions. Thermodynamic parameters and circular dichroism spectra of these oligonucleotides were compared.     

Fatty acid structural requirements for activity of arachidonoyl-CoA synthetase

We have examined the fatty acid substrate specificity of arachidonoyl-CoA synthetase from human platelet membranes. A variety of positional isomers and chain-length analogs of arachidonic acid [20:4(5, 8, 11, 14)] were synthesized, and assayed for their ability to inhibit arachidonoyl-CoA formation or to serve as substrates for the synthetase. The chain-length specificity of the synthetase for delta 8,11,14 trienoic fatty acids was C19 greater than C18 = C20 much greater than C21 greater C22. Inhibition activity by positional isomers of arachidonate was 20:4(5, 8, 11, 14) approximately equal to 20:4(6, 9, 12, 15) = 20:4(7, 10, 13, 16) much greater than 20:4(4, 7, 10, 13), however, Vmax for arachidonate was greater than that for 20:4(6, 9, 12, 15). The enzyme apparently "counts" double bonds from the carboxyl terminus. As counted from the methyl terminus we found that several n-6,-9,-12 fatty acids were ineffective as inhibitors [18:3(6, 9, 12); 19:4)4, 7, 10, 13); 21:3(9, 12, 15)], whereas all methylene-interrupted tri- and tetraenoic fatty acids which contained delta 8 and delta 11 double bonds were potent inhibitors. The delta 11 double bond was best associated with optimal inhibition: 20:3(5, 11, 14) had a lower Ki than 20:3(5, 8, 14). 13-Methyl-20:3(8, 11, 14) did not inhibit the enzyme. Partially purified enzyme from calf brain, depleted of nonspecific long-chain acyl-CoA synthetase, exhibited the same fatty acid specificity as crude platelet enzyme.     

Synthesis and high stability of complementary complexes of N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides.

Two simple methods for the synthesis of oligonucleotides bearing a N-(2-hydroxyethyl)phenazinium (Phn) residue at the 5'- and/or 3'-terminal phosphate groups are proposed. By forming complexes between a dodecanucleotide d(pApApCpCpTpGpTpTpTpGpGpC), a heptanucleotide d(pCpCpApApApCpA), and Phn derivatives of the latter, it is shown that the introduction of a dye at the end of an oligonucleotide chain strongly stabilizes its complementary complexes. The Tmax and the thermodynamic parameters (delta H, delta S, delta G) of complex formation were determined. According to these data, coupling of a dye with the 5'-terminal phosphate group is the most advantageous: delta G(37 degrees C) is increased by 3.59 +/- 0.04 kcal/mol compared to 2.06 +/- 0.04 kcal/mol for 3'-Phn derivatives. The elongation of the linker, which connects the dye to the oligonucleotide, from a dimethylene up to a heptamethylene usually leads to destabilization of the oligonucleotide complex. The complementary complex formed by the 3',5'-di-Phn derivative of the heptanucleotide was found to be the most stable among all duplexes investigated. Relative to the unmodified complex the increase in free energy was 4.96 +/- 0.04 kcal/mol. The association constant of this modified complex at 37 degrees C is 9.5 x 10(6) M-1, whereas the analogous value for the unmodified complex is only 3 x 10(3) M-1.