Inhibition of pancreatic ribonuclease by 2''-5'' and 3''-5'' oligonucleotides.

Forty different oligonucleotides were investigated as possible inhibitors of the depolymerizing activity of RNase A. The strongest inhibitors among the diribonucleoside 2'-5' mono- phosphates were: G2'-5'G, C2'-5'G and U2'-5'G, and among the diribonucleoside 3'-5' monophosphates: ApU, ApC and GpU. Of the eight trinucleotides investigated, ApApUp, ApApCp and ApGpUp were the strongest inhibitors. All four dinucleotides studied (ApUp, ApCp, GpUp and GpCp) were very strong inhibitors, ApUp being the strongest one. The results show that the nature of the various bases in the oligonucleotide has an effect on the degree of inhibition, and that the 3' phosphomonoester group increases the binding of the oligonucleotide to RNase A. These inhibitors can be used in physicochemical and biochemical studies of ribonuclease.     

The preparation and properties of 4-thiouridine containing 2''-5'' and 3''-5'' dinucleoside monophosphates.

2'-5' and 3'-5' dinucleoside monophosphates containing 4-thiouridine were prepared by the thiolation of the cytosine containing compounds and purified by chromatography on a DEAE-Sephadex column. The chromatographic and optical properties of the isomers are compared.     

5''-3'' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis.

The application of T7 and lambda exonuclease to phosphorothioate-based oligonucleotide-directed mutagenesis was investigated. Oligonucleotide primers designed to introduce single or double base mismatches, an insertion or a deletion (each of 16 bases) were annealed to M13 phage derivatives. Double stranded closed circular DNA (RF IV) containing phosphorothioate internucleotidic linkages in the (-)strand was prepared enzymatically from these templates. A nick was introduced into the (+)strand of the hetroduplex DNA. This nicked DNA (RF II) was subjected to treatment with T7 or lambda exonuclease. Both of these enzymes were able to degrade almost all of the viral (+)strand when presented with DNA containing one or two base mismatches. Repolymerisation of the DNA after the gapping reaction, followed by transfection into E. coli cells gave mutational efficiencies of up to 95%. In the case of RF II DNA prepared with insertion or deletion primers these exonucleases could only partially degrade the viral (+)strand but were nevertheless highly efficient in such mutagenesis experiments.     

Association of 2''-5'' oligoribonucleotides.

Oligoribonucleotides with 2'-5' linkages have been synthesized on solid support. UV melting and CD experiments indicate complementary strands associate to give complexes with melting temperatures 30 to 40 degrees C lower than for duplexes formed by 3'-5' oligoribonucleotides with the same sequence. UV melting and imino proton NMR spectra and NOEs for (2'-5') CGGCGCCG are consistent with formation of an antiparallel duplex. The results suggest greater duplex stability was one factor favoring 3'-5' over 2'-5' linkages in evolution.     

Regulation of synthesis of guanosine 3'':5''-cyclic monophosphate in neuroblastoma cells.

The increase in intracellular cyclic GMP concentrations in response to muscarinic-receptor activation in N1E-115 neuroblastoma cells is dependent on extracellular Ca2+ ion. The calcium ionophore A23187 can also evoke an increase in cyclic GMP in the presence of Ca2+ ion. Most (about 85%) of the guanylate cyclase activity of broken-cell preparations is found in the soluble fraction. The soluble enzyme can utilize MnGTP (Km = 55 micrometer), MgGTP (Km = 310 micrometer) and CaGTP (Km greater than 500 micrometer) as substrates. Free GTP is a strong competitive inhibitor (Ki approximately 20 micrometer). The enzyme possesses an allosteric binding site for free metal ions (Ca2+, Mg2+ and Mn2+). The membrane-bound guanylate cyclase is qualitatively similar to the soluble form, but has lower affinity for the metal-GTP substrates. Entry of Ca2+ into cells may increase cyclic GMP concentration by activating guanylate cyclase through an indirect mechanism.     

Chemical synthesis of 5''-pyrophosphate and triphosphate derivatives of 3''-5'' ApA, ApG, GpA and GpG. CD study of the effect of 5''-phosphate groups on the conformation of 3''-5'' GpG.

A simple, two-step method is described for the synthesis of the 5'-pyro- and triphosphate derivatives of 3'-5' ApA, ApG, GpA and GpG. The readily accessible 2'(3')-5' ApA, ApG, GpA and GpG were converted in one step to the corresponding 5'-phosphoramidate derivatives which were then transformed to the 5'-pyro- and triphosphates. CD spectra of 3'-5' pn GpG (n = 0,1,2 or 3) derivatives, measured at pH 1, indicated stabilization of the (syn) G+p (anti)G conformation by the 5'-phosphate groups.     

The effect of (2''-5'') and (3''-5'') phosphodiester linkages on conformational and stacking properties of cytidylyl-cytidine in aqueous solution.

Conformational properties of (2'-5') and (3'-5') CpC have been determined by proton magnetic resonance spectroscopy at 220 MHz. The ribose ring structures are predominantly 3E with the exception of the ring from the 2'-phosphate fragment of C(2'-5')pC which exhibits an 2E pucker. Bases are oriented anti with respect to the ribose and the conformations about C4'-C5', C5'-O5', C3'-O3' (C2'-O2') are gg, g'g', and g+ in equilibrium g-, respectively. The dimers exist as mixtures of stacked (g+g+ and g-g- about the P-O(C) bonds) and unstacked species at 20 degrees C. Stacking is estimated to be 35% in both dimers.     

Preparation of the individual diastereomers of adenylyl-(2''-5'')-P-thioadenylyl-(2''-5'')-adenosine and their 5''-phosphorylated derivatives.

The individual diastereomers of trimer A2'p5'A2'p(s)5'A, containing one phosphorothioate linkage, were prepared via a modified hydroxybenzotriazole phosphotriester approach. The 5'-phosphorylated derivatives of the latter compounds were obtained after phosphorylation with a 6-trifluoromethyl-1-benzotriazolyl activated phosphoromorpholidate.     

Conformational studies of the smallest structural motifs of DNA detectable via vibrational circular dichroism: cytidylyl-(3''-5'')-guanosine and guanylyl-(3''-5'')-cytidine.

The infrared absorption and vibrational circular dichroism (VCD) spectra of buffered aqueous solutions of cytidylyl-(3'-5')-guanosine (5'(CG)3') and guanylyl-(3'-5')-cytidine (5'(GC)3') are reported. Under low ionic strength conditions, these dinucleotides exhibit VCD features that can be predicted qualitatively from structural data of (CG)2 and (GC)2 sequences of poly(dG-dC).poly(dG-dC), using the exciton model for infrared VCD intensities.     

The modified nucleosides of tRNAs. II. Synthesis of 2''-O-methylcytidylyl (3''-5'') cytidine.

The synthesis of 2'-O-methylcytidylyl (3'-5')cytidine by the triester method using as protecting groups, 2,2,2-trichloroethyl for phosphate hydroxyl group, p-chlorophenyoxyacetyl for 5-hydroxyl group, methoxymethylidene for 2',3'-cis-diol system, and benzoyl for the exo-amino group of cytidine is presented. The obtained product was characterised by UV, electrophoresis, chromatography and an enzymatic digestion.     

2'' phosphomonoester, 3''-5'' phosphodiester bond at a unique site in a circular viral RNA.

Solanum nodiflorum mottle virus (SNMV) RNA2 is a single-stranded, covalently closed circular molecule. RNase T2 or nuclease P1 digests of this RNA contain a minor nucleotide of unusual chromatographic and electrophoretic mobility. This nucleotide is resistant to further digestion by T2 or P1 ribonucleases, or by alkali, but is sensitive to venom phosphodiesterase digestion. Alkaline phosphatase digestion yields a product which is RNase T2 and P1 sensitive. The products of these various digests show that the minor nucleotide is a ribonuclease-resistant dinucleotide carrying a 2' phosphomonoester group with the core structure C2'p3'p5'A. This dinucleotide is found in a unique RNase T1 product of SNMV RNA2, thus establishing a unique location in the sequence for the 2' phosphomonoester group at residue 49. Identical results have been obtained with a second related virus. The phosphomonoester group probably results from the RNA ligation event by which the molecules were circularised.     

Repressible extracellular phosphodiesterases showing cyclic 2'',3''- and cyclic 3'',5''-nucleotide phosphodiesterase activities in Neurospora crassa.

Two molecular species of repressible extracellular phosphodiesterases showing cyclic 2',3'- and cyclic 3',5'-nucleotide phosphodiesterase activities were detected in mycelial culture media of wild-type Neurospora crassa and purified. The two molecular species were found to be monomeric and polymeric forms of an enzyme constituted of identical subunits having molecular weights of 50,000. This enzyme had the same electrophoretic mobility as repressible acid phosphatase. The enzyme designated repressible cyclic phosphodiesterase showed pH optima of 3.2 to 4.0 with a cyclic 3',5'-AMP substrate and 5.0 to 5.6 with a cyclic 2',3'-AMP substrate. Repressible cyclic phosphodiesterase was activated by MnCl2 and CoCl2 with cyclic 2',3'-AMP as substrate and was slightly activated by MnCl2 with cyclic 3',5'-AMP. The enzyme hydrolyzed cyclic 3',5'- and cyclic 2',3'-nucleotides, in addition to bis-rho-nitrophenyl phosphate, but not certain 5' -and 3'-nucleotides. 3'-GMP and 3'-CMP were hydrolyzed less efficiently. Mutant strains A1 (nuc-1) and B1 (nuc-2), which cannot utilize RNA or DNA as a sole source of phosphorus, were unable to produce repressible cyclic phosphodiesterase. The wild type (74A) and a heterocaryon between strains A1 and B1 produced the enzyme and showed growth on orthophosphate-free media containing cyclic 2',3'-AMP or cyclic 3',5'-AMP, whereas both mutants showed little or no growth on these media.     

A simple and convenient synthesis of 3''-5''- or 2''-5''-linked oligoribonucleotide by polymerization of unprotected ribonucleoside using phosphorus tris-azole

Oligoribonucleotides have been synthesized directly from unprotected ribonucleosides by a chemical polymerization approach using phosphorus tris-azoles. The procedure involves two steps: (i) the reaction of unprotected ribonucleoside with phosphorus tris-azole and (ii) the in situ oxidation of the resulting phosphite with iodine and water. Several phosphorus tris-azoles were investigated for generating oligoribonucleotide chains. Phosphorus tris-azoles of which azoles are imidazole, 2-methylimidazole, and 2-ethyl-4-methylimidazole were found to be most effective. Uridine, adenosine, and cytidine oligonucleotides were obtained rapidly in high yields without any protection. The inter-ribonucleotidic linkage of the oligomers consists of 3'-5'- and 2'-5'-linkages. The linkage isomers were easily separated by a reverse phase column chromatography. The present approach provides a convenient and potentially useful method for preparing 3'-5'- or 2'-5'-linked oligoribonucleotides.     

Formation of internucleotide 3''-5'' phosphoramidate links by direct coupling of phosphoryl and amino groups.

The stepwise synthesis of oligomers derived from 5'-amino-5'-deoxythymidine 3'-phosphate and 5'-amino-5'-deoxy-3'-O-mono-p-methoxytrityl-thymidine is described. The internucleotide phosphoramidate links were formed by condensation of nucleoside 3'-phosphates with aminonucleosides by means of triphenylphosphine and dipridyl disulfide.     

Polymerization activity of an alpha-like DNA polymerase requires a conserved 3''-5'' exonuclease active site.

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.     

2'',3''=Carbonates in the synthesis of uridine 5''-deoxy and 2'',5''-dideoxy derivatives.

Detritylation of 2',3'-O-carbonyl-5'-O-trityluridine (Ia) with ethereal hydrogen chloride affords 2',3'-O-carbonyluridine (Ib; 83%) which is converted by mesylation to the 5'-mesylcarbonate Ic (75%). Reaction of compound, Ic with tetrabutylammonium bromide in DMF affords the 5'-bromo carbonate Id (77%) which is reduced with tributyltin hydride to the 5'-deoxyuridine 2',3'-cyclic carbonate Ie (70%). When heated with imidazole, compound Ie affords the 2,2'-anhydro derivative IIa (76%) which is converted to the 2'-chloro derivative IIIa (88%) on heating with HC1/DMF. The tributyltin hydride reduction of compound IIIa gives 2',5'-dideoxyuridine (IIIb; 68%). When heated with NaHCO3 in DMF, the 5'-bromo carbonate Id affords the anhydro bromo derivative IIb (50%) which is converted to the 2',5'-dichloro derivative IIIc (86%) on heating with HC1/DMF. The tributyltin hydride reduction of compound IIIc affords the 2',5'-dideoxy derivative IIIb (59%). Alkaline hydrolysis of the 2,2'-anhydro derivative IIa affords the arabinosyl derivative IVa which is converted to the diacetyl derivative IVb (34%) by acetylation. When refluxed in water, the 2',3'-cyclic carbonates Ib, Id, and Ie are hydrolysed to the parent nucleosides, namely, uridine (Va; 81%), 5'-bromo-5'-deoxyuridine (Vb; 78%), and 5'-deoxyuridine (Vc; 83%). Hydrolysis of carbonates Ib and Ie is accompanied by the formation of the 2,2'-anhydro derivatives IIc (10%) and IIa (5%) as by-products.