Automated parallel synthesis of 5′-triphosphate oligonucleotides and preparation of chemically modified 5′-triphosphate small interfering RNA

A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5′-triphosphate and 5′-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5′-triphosphates and 5′-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5′-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5′-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5′-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform.     

Efficient chemoenzymatic synthesis of uridine 5′-diphosphate N-acetylglucosamine and uridine 5′-diphosphate N-trifluoacetyl glucosamine with three recombinant enzymes

Uridine 5′-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a natural UDP-monosaccharide donor for bacterial glycosyltransferases, while uridine 5′-diphosphate N-trifluoacetyl glucosamine (UDP-GlcNTFA) is its synthetic mimic. The chemoenzymatic synthesis of UDP-GlcNAc and UDP-GlcNTFA was attempted by three recombinant enzymes. Recombinant N-acetylhexosamine 1-kinase was used to produce GlcNAc/GlcNTFA-1-phosphate from GlcNAc/GlcNTFA. N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12 MG1655 was used to produce UDP-GlcNAc/GlcNTFA from GlcNAc/GlcNTFA-1-phosphate. Inorganic pyrophosphatase from E. coli K12 MG1655 was used to hydrolyze pyrophosphate to accelerate the reaction. The above enzymes were expressed in E. coli BL21 (DE3) and purified, respectively, and finally mixed in one-pot bioreactor. The effects of reaction conditions on the production of UDP-GlcNAc and UDP-GlcNTFA were characterized. To avoid the substrate inhibition effect on the production of UDP-GlcNAc and UDP-GlcNTFA, the reaction was performed with fed batch of substrate. Under the optimized conditions, high production of UDP-GlcNAc (59.51?g/L) and UDP-GlcNTFA (46.54?g/L) were achieved in this three-enzyme one-pot system. The present work is promising to develop an efficient scalable process for the supply of UDP-monosaccharide donors for oligosaccharide synthesis.     

The synthesis and turnover of 5′-nucleotidase in primary cultured hepatocytes

The synthesis and degradation of 5′-nucleotidase has been studied in rat hepatocytes. Primary cultures of rat hepatocytes were established with the cells showing evidence of polarity after 24–36 h in culture. After a 30 h lag period 5′-nucleotidase activity increased to a plateau level similar to the activity found in whole liver. The half life of the enzyme after reaching the plateau of activity was 22.8 h. Pulse-chase biosynthetic labelling studies of 5′-nucleotidase in the cultured hepatocytes using [³⁵S]methionine showed that the 5′-nucleotidase monomer was synthesised as an M_r 67 000 form which was converted to the mature M_r 72 000 form. [³⁵S]Methionine labelling studies in the presence of tunicamycin showed that the unglycosylated protein monomer was an M_r 57 000 form. The immature M_r 67 000 form of 5′-nucleotidase was sensitive to endoglycosidase H, whereas the mature form was sensitive only to endoglycosidase F. The data presented are consistent with 5′-nucleotidase in a polarised cell being synthesised and processed like other membrane glycoproteins, in contrast to earlier reports.     

Inhibition of the synthesis of polyamines and macromolecules by 5′-methylthioadenosine and 5′-alkylthiotubercidins in BHK21 cells

5′-Methylthioadenosine and four 5′-alkylthiotubercidins were tested for their ability to inhibit polyamine synthesis in vitro and to decrease polyamine concentration and prevent growth of baby-hamster-kidney (BHK21) cells. 5′-Methylthioadenosine and 5′-methylthiotubercidin decreased the activity of spermidine synthase from brain to roughly the same extent, whereas brain spermine synthase was much more strongly inhibited by 5′-methylthioadenosine compared with 5′-methylthiotubercidin. These nucleoside derivatives also inhibited the growth of BHK21 cells and increased the concentration of putrescine. 5′-Methylthioadenosine decreased cellular spermine concentration, whereas 5′-methylthiotubercidin lowered the concentration of spermidine. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were enhanced in cells grown in the presence of 5′-methylthiotubercidin. The growth inhibition produced by these nucleoside derivatives was not reversed by exogenous spermidine or spermine. 5′-Ethylthiotubercidin, 5′-propylthiotubercidin and 5′-isopropylthiotubercidin did not appreciably inhibit spermidine or spermine synthase in vitro or decrease the cellular polyamine content, but effectively prevented the growth of BHK21 cells. All nucleoside derivatives at concentrations of 0.2–1 mm caused a rapid inhibition of protein synthesis. It is concluded that the growth inhibition produced by 5′-methylthioadenosine and 5′-alkylthiotubercidins was not primarily due to polyamine depletion but other target sites, for instance the cellular nucleotide pool, cell membranes etc. must be considered.     

Solid-phase synthesis of 5′-triphosphate 2′-5′-oligoadenylates analogs with 3′-O-biolabile groups and their evaluation as RNase L activators and antiviral drugs

5′-Triphosphate 2′-5′-oligoadenylate (2–5A) is the central player in the 2–5A system that is an innate immunity pathway in response to the presence of infectious agents. Intracellular endoribonuclease RNase L activated by 2–5A cleaves viral and cellular RNA resulting in apoptosis. The major limitations of 2–5A for therapeutic applications is the short biological half-life and poor cellular uptake. Modification of 2–5A with biolabile and lipophilic groups that facilitate its uptake, increase its in vivo stability and release the parent 2–5A drug in an intact form offer an alternative approach to therapeutic use of 2–5A. Here we have synthesized the trimeric and tetrameric 2–5A species bearing hydrophobic and enzymolabile pivaloyloxymethyl groups at 3′-positions and a triphosphate at the 5′-end. Both analogs were able to activate RNase L and the production of the trimer 2–5A (the most active) was scaled up to the milligram scale for antiviral evaluation in cells infected by influenza virus or respiratory syncytial virus. The trimer analog demonstrated some significant antiviral activity.     

The kinetics and feedback inhibition of cytidine 5′-triphosphate synthetase in wild-type and mutant Chinese hamster cells

The kinetics and cytidine 5-triphosphate (CTP) feedback inhibition of CTP synthetase in wild-type and four mutants of Chinese hamster V79 cells have been studied. The enzymes of the wild type and three of the four mutants exhibited positive cooperativity with the substrate uridine 5-triphosphate (UTP). Three of the mutants had K _{m app} and S ₅₀ valuves distinctly greater than those of the wild type, while the fourth mutant had values similar to those of the wild type. all four mutants exhibited resistance to CTP feedback inhibition, while the wild type was sensitive to such inhibition. It is postulated that a single mutational event in each mutant had caused a concomitant change of the enzyme in its binding both to the substrate UTP and to the end-product CTP.This work was supported by Grant GM 20608 from the U.S. Public Health Service.     

The addition of 5′ cap structures occurs early in hnRNA synthesis and prematurely terminated molecules are capped

After cells were labeled by brief exposure to ³H-methyl-L-methionine, the majority of labeled 5′ terminal cap I (m⁷GpppN₁mpN₂p) oligonucleotide structures were in nuclear RNA (hnRNA) molecules ～750 nucleotides or less in length. After longer label times, the proportion of cap I structures in nuclear molecules longer than mRNA rose to approximately 60% of the total, but approximately 40% of the cap I structures were still in molecules shorter than ～750 nucleotides. The cap I structures in both long and short hnRNA chains contained all four 2′ methylated nucleotides in the N₁ position in about the same proportion as in mRNA. None of the large hnRNA molecules could be demonstrated to contain 5′ pppXp termini; the only such terminus in high molecular weight RNA was pppAp which was decreased markedly by low doses of actinomycin and is presumably the terminus of pre-rRNA. These results raise the possibilities that hnRNA chains can initiate with any of the four nucleotides, that capping occurs very close to or at the start of hnRNA chain synthesis and that approximately 40% of the hnRNA chains may be prematurely terminated.     

Stress-induced formation of echinatin and a metabolite, 5′-prenyl-licodione,in cultured Glycyrrhiza echinata cells

The production of a retrochalcone, echinatin, by isoflavone-rich Glycyrrhiza echinata (M-2) cultured cells was stimulated by the addition of yeast extract or calcium alginate beads to the culture medium. Combined addition of yeast extract and cycloheximide suppressed the formation of retrochalcone, suggesting de novo synthesis. A new metabolite was isolated from the induced cells and its structure was determined to be 1-[2,4-dihydroxy-5-(3-methyl-2-butenyl)phenyl]-3-(4-hydroxyphenyl)-1,3-propanedione (5′-prenyl-licodione).     

Synthesis, characterization and properties of uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)

1. A method was developed for synthesizing UDP-apiose [uridine 5'-(alpha-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5'-(alpha-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5'-(alpha-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [(3)H]UDP-[U-(14)C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the (3)H/(14)C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100 degrees C for 15min; (b) degraded at pH8.0 and 100 degrees C for 3min; (c) used as a substrate in the enzymic synthesis of [(14)C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [(3)H]UDP-[U-(14)C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-(14)C]apiose and phosphate formed on alkaline degradation of UDP-[U-(14)C]apiose was alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-(14)C]apiose and phosphate formed on acid hydrolysis of alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-(14)C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-(14)C]apiose to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80 degrees C, at pH8.0 and 25 degrees C and at pH8.0 and 4 degrees C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-(14)C]-apiose to d-[U-(14)C]apiose and UDP at pH3.0 and 40 degrees C was 4.67min. After 20 days at pH6.2-6.6 and 4 degrees C, 17% of the starting UDP-[U-(14)C]apiose was degraded to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-(14)C]apiose and UDP. After 120 days at pH6.4 and -20 degrees C 2% of the starting UDP-[U-(14)C]apiose was degraded and 4% was hydrolysed.     

Different effects of thymidine and 5-fluorouracil 2′-deoxyriboside on biosynthetic events in cultured P815Y mast cells

1. Addition of 2mm-thymidine, although resulting in the cessation of cell division, allows the continuation of phospholipid and protein synthesis and results in an increase in mean cell volume for at least 15h. 2. 5-Fluorouracil 2'-deoxyriboside inhibits cell division but differs from thymidine by inhibiting the synthesis of phospholipid and protein in a more marked manner. 3. The relation between these results and the P815Y cell cycle is discussed.     

The synthesis and antituberculosis activity of 5′-nor carbocyclic uracil derivatives

A series of new carbocyclic uracil derivatives were synthesized and evaluated as potential antituberculosis agents. Racemic 1-[4′-hydroxy-2′-cyclopenten-1′-yl]-5-tetradecynyluracil completely inhibited the growth of Mycobacterium tuberculosis H37Rv in vitro at a concentration of 10 μg/ml. Individual (+) and (?) isomers of the above uracil derivative were isolated and showed the same level of activity against two strains of Mycobacterium tuberculosis: laboratory sensitive (H37Rv) and clinical resistant to five top antituberculosis drugs (MS-115).     

Adenosine 3′:5′-cyclic monophosphate concentrations and phosphodiesterase activities during axenic growth and differentiation of cells of the cellular slime mould Dictyostelium discoideum

During growth of myxamoebae of Dictyostelium discoideum (strain Ax-2) in axenic medium, the myxamoebae secrete cyclic AMP. As the cells leave the exponential phase of growth and enter the stationary phase, there is an approximate doubling of the intracellular cyclic AMP content, but the amount of extracellular cyclic AMP remains proportional, at all times, to the number of myxamoebae present. During development of axenically grown myxamoebae, there is first a rise in the intracellular concentration of cyclic AMP, followed by a rise in the amount of extracellular cyclic AMP, which reaches a peak at the time of aggregation and then declines. There is a second peak in the amount of extracellular cyclic AMP found at the time of fruiting-body formation, but this second peak is not associated with a rise in the intracellular cyclic AMP concentration. Controls thus exist over the synthesis and secretion of cyclic AMP. Evidence is presented for the belief that the activity of the adenylate cyclase enzyme controls the amount of cyclic AMP synthesized rather than the activity or amount of cyclic AMP phosphodiesterase present. Similar changes occur in extracellular cyclic AMP and phosphodiesterase concentrations during incubation of myxamoebae in buffered suspensions to those occuring during the first few hours of development of such cells on solid media, but the timing of these changes is different.     

The rapid transient stimulation of both cytoplasmic and mitochondrial ornithine decarboxylases in the liver of thyroidectomized rats by 3,5,3′-triiodo-L-thyronine

Evidence is presented that liver from thyroidectomized rats has ornithine decarboxylase (ODC) in both mitochondrial and soluble fractions. The cytosolic activity was stimulated 7-fold and the mitochondrial activity 3-fold 15 min after the administration of triiodothyronine (T₃) . While the rapid transient stimulation of ODC could represent a direct intracellular response to T₃, an incidental effect on a very-rapidly-turning-over enzyme seems more likely; this suggests thatin vivo ODC may be controlled by a demand for polyamines.     

The effect of ethylene and cytokinin on guanosine 5′-triphosphate binding and protein phosphorylation in leaves of Arabidopsis thaliana

Binding of [α-³²P]guanosine 5′-triphosphate ([α-³²P]GTP) has been demonstrated in a Triton X-100-solubilised membrane fraction from leaves of Arabidopsis thaliana (L.) Heynh. Binding was stimulated by 1 h pre-treatment of leaves with ethylene and this effect was antagonised by the inclusion of N⁶-benzyladenine in the medium used for homogenisation. The ethylene-insensitive mutants eti5 and etr showed contrasting responses. In eti5 the constitutive level of GTP binding was higher than in the wild type whereas in etr the level was much lower. Neither ethylene nor cytokinin affected GTP binding in the mutants. The GTP-binding activity was localised in two bands at 22 and 25 kDa, both of which were immunoprecipitated by anti-pan-Ras antibodies, indicating that the activity is due to small GTP-binding proteins. In a similar membrane fraction, ethylene was shown to increase protein phosphorylation and benzyladenine antagonised this effect. In eti5 the constitutive level of protein phosphorylation was higher than in the wild type, but benzyladenine increased activity substantially while ethylene was without effect. In etr, protein phosphorylation was lower than in the wild type, ethylene was without effect, but cytokinin increased activity. A protein of M_r 17 kDa was detected on gels using antibodies to nucleoside diphosphate kinase. Phosphorylation of this protein was upregulated by ethylene but nucleoside diphosphate kinase activity was unaffected. The results are compared with the effect of the two hormones on the senescence of detached leaves and discussed in relation to pathways proposed for ethylene signal transduction. Received: 23 November 1998 / Accepted: 10 December 1998     

A 31P-NMR study of mono- and dimagnesium complexes of adenosine 5′-triphosphate and model systems

³¹P-NMR chemical shifts and spin-lattice relaxation times of ATP (adenosine 5′-triphosphate), ribose 5′-triphosphate and tripolyphosphate show closely similar behaviour in aqueous solution at pH 7.5 on titration with Mg²⁺. The results are interpreted in terms of formation of 1 : 1 and 2 : 1 (dimagnesium) complexes with Mg²⁺ bound exclusively to the triphosphate chain. Stability constants for these complexes are reported. It is suggested that the predominant form of the 1 : 1 complexes has Mg²⁺ bound in tridentate manner (via non-bridging oxygen) to the α, β and γ phosphorus atoms; whilst that of the 2 : 1 complexes has each Mg²⁺ bound in bidentate manner, one to the α and β, and the other to the β and γ, phosphorus positions.     

Differential effects of ribosomal proteins and Mg2+ ions on a conformational switch during 30S ribosome 5′-domain assembly

Ribosomal protein S4 nucleates assembly of the 30S ribosome 5′ and central domains, which is crucial for the survival of cells. Protein S4 changes the structure of its 16S rRNA binding site, passing through a non-native intermediate complex before forming native S4-rRNA contacts. Ensemble FRET was used to measure the thermodynamic stability of non-native and native S4 complexes in the presence of Mg²⁺ ions and other 5′-domain proteins. Equilibrium titrations of Cy3-labeled 5′-domain RNA with Cy5-labeled protein S4 showed that Mg²⁺ ions preferentially stabilize the native S4-rRNA complex. In contrast, ribosomal proteins S20 and S16 act by destabilizing the non-native S4-rRNA complex. The full cooperative switch to the native complex requires S4, S16, and S20 and is achieved to a lesser degree by S4 and S16. The resulting thermodynamic model for assembly of the 30S body illustrates how ribosomal proteins selectively bias the equilibrium between alternative rRNA conformations, increasing the cooperativity of rRNA folding beyond what can be achieved by Mg²⁺ ions alone.     

Adsorption of 5′-AMP and catalytic synthesis of 5′-ADP onto phosphate surfaces: Correlation to solid matrix structures

A non-enzymatic formation of 5-ADP starting from phosphorylation of 5-AMP in the presence of either calcium phosphate or calcium pyrophosphate precipitates is reported. This reaction is taken as a model for the study of heterogeneous catalysis of transphosphorylation in prebiotic conditions. Experiments were performed in completely aqueous media and in media containing dimethyl sulfoxide (Me₂S0), to simulate periods of dehydration in primitive aquatic environments. It has been observed that the nucleotide is adsorbed onto both calcium phosphate and calcium pyrophosphate in accordance with Langmuir isotherms. Adsorptive capacity and affinity of the precipitates for nucleotide are changed by the presence of Me₂SO, suggesting that the interaction between biomonomers and surfaces can be modulated by the degree of hydration of the anionic components of these compounds. In completely aqueous environments, formation of 5-ADP from 5-AMP adsorbed on precipitates of calcium phosphate and calcium pyrophosphate is very small. However, in the presence of 60% Me₂SO this synthesis increases by factors of 3 and 6 for surfaces of calcium phosphate and calcium pyrophosphate, respectively, and follows first-order kinetics. Determinations of free energy changes show that phosphorylation of 5-AMP adsorbed to these precipitates is thermodynamically favorable. Depending on the precipitation time of the samples and the composition of the medium, structural analysis of these precipitates by electron and X-ray diffraction shows changes in their cristallinity grade. It is proposed that these changes are responsible for the modulation of the quantity of adsorbed nucleotides to the surface of solid matrices as well as the catalytic activity of the precipitates.Abbreviations 5-AMP 5-adenosine monophosphate - 5-ADP 5-adenosine diphosphate - BTP l,3-bis[tris(hydroxymethyl)-methylamino]propane - CTEM conventional transmission electron microscopy - Tris tris(hydroxymethyl)aminomethane - Pi (H₂PO ₄ ^– /HPO ₄ ^2– ) orthophosphate - Pi.Ca calcium phosphate - PPi (H₃P₂O ₇ ^– /H₂P₂O ₇ ^2– ) pyrophosphate - PPi.Ca calcium pyrophosphate - EGTA [ethylenebis(oxyethylene)nitrilo]tetraacetic acid This work has been submitted to the Department of Biochemistry, Institute of Biomedical Sciences, UFRJ, by A.C.T. in partial fulfillment of requirements for the MS degree.