Nematocidal Action of Microorganisms

The distribution in microorganisms of extracellular enzymes which degrade DNA into deoxymononucleotides was studied. The degradation products of DNA were determined by using 5′-nucleotidase and prostatic nonspecific phosphomonoesterase. It was found that except Bacillus subtilis IFO 3302, the microorganisms which produced the enzymes that catalyze the degradation of RNA into 5′-mononucleotides, produced the enzymes capable of hydrolyzing DNA into 5′-deoxymononucleotides, whereas the microorganisms which produced such enzymes that degrade RNA into 3′-mononuclcotides did not generally produce the enzymes which hydrolyze DNA.     

Degradation of Nucleic Acids and their Related Compounds by Microbial Enzymes

Aspergillus quercinus (IFO 4363) was selected as the most suitable strain to produce 5′-mononucleotides from RNA among several species of Aspergillus which produced enzymes capable of degrading RNA into 5′-mononucleotides.

Aspergillus quercinus produced two kinds of RNA-depolymerases (designated as RNA-deploymerase I and II), phosphodiesterase, phosphomonoesterase and adenylic deaminase in the culture medium. The optimum pH of each enzyme was found to be about 4.5, 7.0, 5.0, 6.0 and 5.5, respectively. Maximal production of these enzymes in the culture medium occurred at 96, 96, 216, 168 and 264 hour culture, respectively. The culture filtrate of Aspergillus quercinus degraded RNA into 3′-mononucleotides at the pH lower than 6.0, into 5′-mono-nucleotides at the pH higher than 8.5 and into both mononucleotides at the pH range between 6.0 and 8.5. 5′-Inosinic acid was prepared from RNA by using the extra- and intracellular enzymes of Aspergillus quercinus.     

Degradation of Nucleic Acids and their Related Compounds by Microbial Enzymes

Seven strains of microorganisms selected by the previous screening tests were further compared on their ability to produce extracellular enzyme systems capable of degrading RNA into 5′-ribonucleotides. As a result, two strains of Streptomyces were finally concluded to be most preferable. When these two were applied, the rate of 5′-nucleotide production reached up to 70%.

Bacillus subtilis was outstanding in its activity to degrade RNA, but its PDase activity producing 5′-nucleotides from RNA was found to be lower than those of Streptomyces strains. A pathway involving 3′- and 5′-nucleotides as intermediates was proposed for the degradation of RNA by the Bacillus enzyme system. The activity of RNA-degrading enzyme system of Bacillus subtilis contained in the supernatant of culture fluid was found to be lost at 700°C but remained to certain extent at 100°C, a possible mechanism for the phenomenon being discussed. Usability of the Bacillus enzyme system in the practical production of 5′-nucleotides under the condition of high RNA concentration was discussed.     

Purification of 5′-O-Trityl-on Oligoribonucleotides. Investigation of Phosphate Migration During Purification and Detritylation.

Abstract

Synthetic oligoribonucleotides (RNA) are efficiently prepared with 2′-O-tert-butyldimethylsilyl nucleoside 3′-O-phosphoramidites with labile base-protection; A_dmf or A_Pac, G_dmf, C_ibu, U. After cleavage from the polystyrene support, the exocyclic amine protecting groups are removed with conc. NH₄OH: ethanol/3:1 by heating at 55°C for 3–5 h. The 2′-O- silyl protecting groups are removed with tetra-n-butylammonium fluoride in THF or more conveniently with neat triethylamine trihydrofluoride. To gain the advantages of increased capacity on reverse phase HPLC and the convenience of cartridge based purification (OPC, Oligonucleotide Purification Cartridge), the 5′ trityl was left on the RNA as the final protecting group to be removed. The mild conditions which are effective for trityl removal are shown to preserve 3′-5′ phosphate linkage integrity in RNA. The absence of phosphate migration is demonstrated by model studies, utilizing N⁴ -isobutyryl-5′-O-DMT-3′-O-TBDMS-2′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) as a control monomer and digestion by 3′-5′ selective P1 nuclease and alkaline phosphatase and HPLC analysis. Oligoribonucleotides were analyzed by Microgel capillary electrophoresis, anion-exchange HPLC, and the enzymatic digest/HPLC method.

     

Adenylyl-(5′, 2′)-adenosine 5′-Phosphate ((2′-5′) pA-A) in Crude Crystals of 5′-Adenylic Acid Isolated from Nuclease P1 (Penicillium Nuclease) Digest of Technical Grade Yeast RNA

Adenylyl (5′,2′)-adenosine 5′-phosphate ((2′-5′)pA-A) was detected in crude crystals of 5′-AMP prepared from Penicillium nuclease (nuclease P₁) digest of a technical grade yeast RNA. While (3′–5′)A-A was split by nuclease P₁, spleen phosphodiesterase, snake venom phosphodiesterase or alkali, (2′–5′)A-A was not split by a usual level of nuclease P₁ or spleen phosphodiesterase. Nuclease P₁ digests of 12 preparations of technical grade yeast RNA tested were confirmed to contain (2′–5′)pA-A. Its content was about 1 to 2% of the AMP component of each RNA preparation. As poly(A) was degraded completely by the Penicillium enzyme into 5′-AMP without formation of any appreciable amount of (2′–5′)pA-A, the technical grade RNA is supposed to contain 2–5′ phosphodiester linkages in addition to 3′–5′ major linkages.     

Synthesis of the Oligoribonucleotides Incorporating 8-Oxo-Guanosine and Evaluation of their Base Pairing Properties

6-O-7-N-Bis(diphenylcarbamoyl)-2-N-phenoxyacetyl-5′-O-dimethoxytrityl-2′-O-{[(triisopropyl- silyl)oxy]methyl}-8-oxoguanosine-3′-yl-β-cyanoethyl-N,N-diisopropylphosphoramidite (5) was synt- hesized as a new phosphoramidite precursor unit for the synthesis of RNA. Compound 5 was successfully incorporated into the middle of the RNA sequences, and the synthesized RNAs were identified by MALDI-TOF mass measurements. Their properties were evaluated for formation of the RNA duplex and RNA/DNA heteroduplex. ORNs 1 and 4 containing 8-oxo-G can form base pairs with rC or dC in an anti conformation, while it can also interact with rA or dA in a syn conformation in the RNA duplex or RNA/DNA heteroduplex. The described synthetic method is therefore a useful procedure for the synthesis of ORN containing 8-oxo-G and contributes to the study of 8-oxo-G in RNA.     

Synthesis of Modified RNA-Oligonucleotides for Structural Investigations

Abstract

RNA exhibits a higher structural diversity than DNA and is an important molecule in biology of life. It shows a number of secondary structures such as duplexes, hairpin loops, bulges, internal loops etc. However, in natural RNA, bases are limited to the four predominant structures U, C, A, and G and so the number of compounds that can be used for investigation of parameters of base stacking, base pairing and hydrogen bond, is limited. We synthesized different fluoromodifications of RNA building blocks: 1′-deoxy-1′-(2,4,6-trifluorophenyl)-ß-D-ribofuranose (F), 1′-deoxy-1′-(2,4,5-trifluorophenyl)-ß-D-ribofuranose (M) and 1′-deoxy-1′-(5-trifluoromethyl-1H-benzimidazol-1-yl)-ß-D-ribofuranose (D). Those amidites were incorporated and tested in a defined A, U- rich RNA sequence (12-mer, 5′-CUU UUC XUU CUU-3′ paired with 3′-GAA AAG YAA GAA-5’) (Schweitzer, B.A.; Kool, E.T. Aromatic nonpolar nucleosides as hydrophobic isosters of pyrimidine and purine nucleosides. J. Org. Chem. 1994, 59, 7238 pp.). Only one position was modified, marked as X and Y respectively. UV melting profiles of those oligonucleotides were measured.     

Oligonucleotide N3′→P5′ Phosphoramidates and Thio‐Phoshoramidates as Potential Therapeutic Agents

Nucleic acids analogues, i.e., oligonucleotide N3′→P5′ phosphoramidates and N3′→P5′ thio‐phosphoramidates, containing 3′‐amino‐3′‐deoxy nucleosides with various 2′‐substituents were synthesized and extensively studied. These compounds resist nuclease hydrolysis and form stable duplexes with complementary native phosphodiester DNA and, particularly, RNA strands. An increase in duplexes' melting temperature, ΔT_m, relative to their phosphodiester counterparts, reaches 2.2–4.0° per modified nucleoside. 2′‐OH‐ (RNA‐like), 2′‐O‐Me‐, and 2′‐ribo‐F‐nucleoside substitutions result in the highest degree of duplex stabilization. Moreover, under close to physiological salt and pH conditions, the 2′‐deoxy‐ and 2′‐fluoro‐phosphoramidate compounds form extremely stable triple‐stranded complexes with either single‐ or double‐stranded phosphodiester DNA oligonucleotides. Melting temperature, T_m, of these triplexes exceeds T_m values for the isosequential phosphodiester counterparts by up to 35°. 2′‐Deoxy‐N3′→P5′ phosphoramidates adopt RNA‐like C3′‐endo or N‐type nucleoside sugar‐ring conformations and hence can be used as stable RNA mimetics. Duplexes formed by 2′‐deoxy phosphoramidates with complementary RNA strands are not substrates for RNase H‐mediated cleavage in vitro. Oligonucleotide phosphoramidates and especially thio‐phosphoramidates conjugated with lipid groups are cell‐permeable and demonstrate high biological target specific activity in vitro. In vivo, these compounds show good bioavailability and efficient biodistribution to all major organs, while exerting acceptable toxicity at therapeutically relevant doses. Short oligonucleotide N3′→P5′ thio‐phosphoramidate conjugated to 5′‐palmitoyl group, designated as GRN163L (Imetelstat), was recently introduced as a potent human telomerase inhibitor. GRN163L is not an antisense agent; it is a direct competitive inhibitor of human telomerase, which directly binds to the active site of the enzyme and thus inhibits its activity. This compound is currently in multiple Phase‐I and Phase‐I/II clinical trials as potential broad‐spectrum anticancer agent.     

Ribonuclease II preserves chloroplast RNA homeostasis by increasing mRNA decay rates,and cooperates with polynucleotide phosphorylase in 3′ end maturation

Ribonuclease R (RNR1) and polynucleotide phosphorylase (cpPNPase) are the two known 3′→5′ exoribonucleases in Arabidopsis chloroplasts, and are involved in several aspects of rRNA and mRNA metabolism. In this work, we show that mutants lacking both RNR1 and cpPNPase exhibit embryo lethality, akin to the non‐viability of the analogous double mutant in Escherichia coli. We were successful, however, in combining an rnr1 null mutation with weak pnp mutant alleles, and show that the resulting chlorotic plants display a global reduction in RNA abundance. Such a counterintuitive outcome following the loss of RNA degradation activity suggests a major importance of RNA maturation as a determinant of RNA stability. Detailed analysis of the double mutant demonstrates that the enzymes catalyze a two‐step maturation of mRNA 3′ ends, with RNR1 polishing 3′ termini created by cpPNPase. The bulky quaternary structure of cpPNPase compared with RNR1 could explain this activity split between the two enzymes. In contrast to the double mutants, the rnr1 single mutant overaccumulates most mRNA species when compared with the wild type. The excess mRNAs in rnr1 are often present in non‐polysomal fractions, and half‐life measurements demonstrate a substantial increase in the stability of most mRNA species tested. Together, our data reveal the cooperative activity of two 3′→5′ exoribonucleases in chloroplast mRNA 3′ end maturation, and support the hypothesis that RNR1 plays a significant role in the destabilization of mRNAs unprotected by ribosomes.     

5'‐ and 3'‐sequences of satellite tobacco necrosis virus RNA promoting translation in tobacco

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5′ cap and a 3′ poly(A) tail. The STNV trailer contains an autonomous translational enhancer domain (TED) that promotes translation in vitro by more than one order of magnitude when combined with the 5′-terminal 173 nt of STNV RNA. We now show that the responsible sequence within the 5′ region maps to the first 38 nt of the STNV RNA. Mutational analysis indicated that the primary sequence of the STNV 5′ 38 nt and TED is important for translation stimulation in vitro, but did not reveal a role for the complementarity between the two. Translation of chimeric STNV-cat RNAs in tobacco protoplasts showed that TED promotes translation in vivo of RNAs lacking a cap and/or a poly(A) tail. Similar to in vitro, TED-dependent translation in tobacco was stimulated further by the STNV 5′ 38 nt.     

Osservazioni Preliminari sui Sistemi Degradanti dell'RNA nei semi di Ricino

Abstract

Preliminary observations on the enzymatic degradation of RNA in castor bean seeds. — Cocucci, Maggio, Monroy and Marrè have shown the decrease of RNA content during ripening in castor bean seeds, and its increase during germination. Furthermore, these Authors have demonstrated that in the dry ripe seeds the ribosomes are undetectable, and that they increase rapidly during germination. Two peaks of ribosomes are easily detected upon ultracentrifugal analysis in germinating seeds (Cocucci and Sturani). These observations were the basis for our investigations of the enzymes of RNA metabolism in castor bean seeds. This paper deals with our preliminary observations on RNA degrading enzymes in these tissues. We have been able to measure RNase activity, phosphodiesterase, 3′-,5′- and 2′-nucleotidases in castor bean seeds at different stages of development. RNase activity (measured in crude extracts) changes little during the ripening process, its rate corresponding to 40–50 μMoles of nucleotides liberated from RNA per hour and per gram of fresh weight. In the dry seeds, RNase activity is 30–40 μMoles of nucleotides/h.g.f.w., and it increases to about 60–70 μMoles/h/g.f.w. after 72 hours of germination.

Phosphodiesterase activity is about 4–5 μMoles/h.g.f.w.

The following rates have been found in seeds almost completely ripe seeds for 3′-, 5′- and 2′-nucleotidase activities, respectively 45–50 μMoles/h.g.f.w.; 6–7 μMoles/h.g.f.w.; 8 μMoles/h.g.f.w.; ATP-ase activity was of about 80–100 μMoles of phosphate liberated /h.g.f.w. - The high activity of 3′-nucleotidase, of the same order of that of RNase, suggests that these two enzymes are responsible for degradation of RNA to nucleosides and inorganic phosphate. Further investigations are being carried on to define the biochemical properties of castor bean RN-ase.     

Small stable RNA maturation and turnover in Bacillus subtilis

Stable RNA maturation is a key process in the generation of functional RNAs, and failure to correctly process these RNAs can lead to their elimination through quality control mechanisms. Studies of the maturation pathways of ribosomal RNA and transfer RNA in Bacillus subtilis showed they were radically different from Escherichia coli and led to the identification of new B. subtilis‐specific enzymes. We noticed that, despite their important roles in translation, a number of B. subtilis small stable RNAs still did not have characterised maturation pathways, notably the tmRNA, involved in ribosome rescue, and the RNase P RNA, involved in tRNA maturation. Here, we show that tmRNA is matured by RNase P and RNase Z at its 5′ and 3′ extremities, respectively, whereas the RNase P RNA is matured on its 3′ side by RNase Y. Recent evidence that several RNases are not essential in B. subtilis prompted us to revisit maturation of the scRNA, a component of the signal recognition particle involved in co‐translational insertion of specific proteins into the membrane. We show that RNase Y is also involved in 3′ processing of scRNA. Lastly, we identified some of the enzymes involved in the turnover of these three stable RNAs.     

Studies on Vitamin B6 Metabolism in Microorganisms

Escherichia freundii alkaline phosphatase was found in a membrane fraction and was purified by procedures involving spheroplast formation with lysozyme and EDTA, and DEAE-cellulose and Sephadex G-150 column chromatographies. Then this enzyme along with other phosphatases was investigated on the ability to transfer the phosphoryl group from p-nitrophenyl phosphate to pyridoxine. It was found that the ability of the transphosphorylation varied with these phosphatases. The transphosphorylation to hydroxy compounds such as alcohols, sugars and nucleosides was also compared. Escherichia freundii acid phosphatase showed the highest activity of transphosphorylation among phosphatases tested. The mechanism of transphosphorylation was discussed.

An enzyme, pyridoxamine 5′-phosphate transaminase, was purified from the cell-free extract of Clostridium kainantoi. The purification procedures involved ammonium sulfate fractionation, protamine sulfate treatment and, DEAE-cellulose, hydroxylapatite, DEAE-Sephadex and Sephadex G-200 column chromatographies. The purified enzyme, which had approximately 2700-fold higher specific activity over the original extract, showed a single schlieren pattern in the ultracentrifuge. From the spectral analysis, it seemed that pyridoxamine 5′-phosphate transaminase did not contain pyridoxal 5′-phosphate as a prosthetic group. It was recognized that the transamination was accelerated by the addition of amino acid and was inhibited by diisopropyl phosphofluoride. Glutamic acid formed in the reaction was identified to be a D-isomer. A study on the substrate specificity showed that the enzyme might be possible to be specific for pyridoxamine 5′-phosphate.

The extracellular formation of vitamin B₆ was searched in marine and terrestrial microorganisms. Two bacterial strains were selected and were identified as Vibrio and Flavobacterium, respectively. Marine microorganisms showed the considerable formation of vitamin B₆ and the presence of vitamin B₆ in sea water was also recognized. The cultural and reaction conditions for vitamin B₆ formation by these strains were investigated. Glycerol was commonly the most effective compound on vitamin B₆ formation among the compounds tested. It was suggested that both bacteria did not have the control system on vitamin B₆ biosynthesis by the amount of possible end products.     

Design and Synthesis of Novel Carbocyclic Versions of 2′-spirocyclopropyl Ribonucleosides as Potent Anti-HCV Agents

The discovery of 2′-spirocyclopropyl-ribocytidine as a potent inhibitor of RNA synthesis by NS5B (IC₅₀ = 7.3 μM), the RNA polymerase encoded by hepatitis C virus (HCV), has led to the synthesis and biological evaluation of carbocyclic versions of 2′-spiropropyl-nucleosides from cyclopentenol 6. Spirocyclopropylation of enone 7 was completed by using (2-chloroethyl)-dimethylsulfonium iodide and potassium t-butoxide to form the desired intermediate 9a. The synthesized nucleoside analogues, 18, 19, 26, and 27, were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line. The synthesized cytosine nucleoside 19 showed moderate anti-HCV activity (IC₅₀ = 14.4 μM).     

Studies on Vitamin B6 Metabolism in Microorganisms

A new phosphotransferring reaction which phosphorylated pyridoxine through the phosphoryl group transfer from p-nitrophenylphosphate was found, and the distribution of the reaction in several microorganisms was investigated. The transferring activity was widely distributed in various kinds of microorganisms, especially in fungi belonging to genus such as Aspergillus. The phosphorylated product was isolated from the reaction mixture with the dried cells of Aspergillus flavus and identified as pyridoxine 5′-phosphate.     

Studies on the “Salvage” Synthesis of Ribonucleosides and Their 5′-Phosphates by Microorganisms

Studies on the “salvage” synthesis of ribonucleosides and their 5′-phosphates from nucleic acid bases by microorganisms were undertaken. After screening test of less than one hundred strains of type culture, it was found that inosine was produced from hypoxanthine by Arthrobacter ureafaciens, A. simplex, Flavobacterium aquatile and F. suaveolens.

In certain conditions, inosine was further oxidized and hydrolyzed into xanthosine, uric acid and etc.

As for the conditions of cultivation and reaction, the components of the medium and pH of the culture medium were important factors.

Using the standard method, the yield of inosine from hypoxanthine by F. suaveolens reached more than 60%, and the conversion was stoichiometric and any other by-products were not detected.

Inosine, xanthosine, guanosine and uridine were produced from adenine, xanthine, guanine and uracil, respectively, by F. suaveolens.     

Conformational Study of DNA-RNA Duplexes Containing MMI Substituted Phosphodiester Linkages by FTIR Spectroscopy

Abstract

Six methylene(methylimino) (MMI, Bhat et al. J. Org. Chem., 61, 8186, 1996) linked oligonucleotides a-f (* = MMI linkage; 5′-GCGT*TT*TT*TT*TT*TGCG-3′) containing various combinations of 2′-O-methyl and 2′-fluoro substituent were synthesized as a model to study the global conformational change upon hybridization to the complement RNA. Fourier transform infrared (FTIR) spectroscopic technique has been used to study and compare the influence of these modifications on the solution conformation of 2′-modified MMI DNA-RNA duplexes. FTIR analysis of the single-stranded RNA (5′-CGCAAAAAAAAAACGC-3′) and the modified oligonucleotides a-f showed that all sugar residues adopted a C3′-endo conformation (North-type). Stable duplexes were formed when oligonucleotides a-f were hybridized to the complement RNA. These duplexes retained the original C3′-endo conformation for all sugar residues, hallmark of an A-form of duplex. We postulate that the observed preorganization of the sugar residues and oligonucleotides containing 2′-modified MMI modifications may play an important role in both improving the recognition of RNA target and enhancing the stability of duplex formation with RNA.     

Partial characterization of 5′(3′)-ribonucleotide and 3′-ribonucleotide phosphohydrolases from Tradescantia albiflora leaves

Two acid phosphomonoesterases, 5′(3′)-ribonucleotide phosphohydrolase and 3′-ribonucleotide phosphohydrolase, were isolated from Tradescantia albiflora leaf tissue and purified by ammonium sulphate precipitation, gel filtration on Sephadex G-200 and repeated chromatography on DEAE-cellulose. The enzymes differed in their sensitivity to dialysis against 1 mM EDTA; the activity of 5′(3′)-ribonucleotide phosphohydrolase was unaffected, while 3′-ribonucleotide phosphohydrolase showed an increase of 60–90%. Both enzymes were rapidly inactivated above 50°. Their ion sensitivity was identical: 1 m M Zn²⁺ and Fe²⁺ were inhibitors for both by 20–80%; while Mg²⁺, Ca²⁺, Co²⁺, K⁺, Na⁺ at 1–10 mM had no significant effect on the activity of either enzyme. Inorganic phosphate inhibited both enzymes almost completely. EDTA (1 mM) did not inhibit either enzyme; none of the divalent cations tested were enzyme activators. 3′-Ribonucleotide phosphohydrolase hydrolysed both 3′- and 5′-nucleoside monophosphates (3′-AMP, 3′-CMP, 3′-GMP, 3′-UMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP). 5′(3′)-Ribonucleotide phosphohydrolase showed a preference for the 3′-nucleoside monophosphates. Adenosine 3′,5′-cyclic monophosphate, purine and pyrimidine 2′,3′-cyclic mononucleotides at 0.1–1.OmM did not inhibit the enzymes.