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At least 199 records · Page 11Linked to original sources
PubMed1990

Structure of 11-deoxydaunomycin bound to DNA containing a phosphorothioate.

The anthracyclines form an important family of cancer chemotherapeutic agents with a strong dependence of clinical properties on minor differences in chemical structure. We describe the X-ray crystallographic solution of the three-dimensional structure of the anthracycline 11-deoxydaunomycin plus d(CGTsACG). In this complex, two drug molecules bind to each hexamer duplex. Both the drug and the DNA are covalently modified in this complex in contrast with the three previously reported DNA-anthracycline complexes. In the 11-deoxydaunomycin complex the 11 hydroxyl group is absent and a phosphate oxygen at the TpA step has been replaced by a sulfur atom leading to a phosphorothioate with absolute stereochemistry R. Surprisingly, removal of a hydroxyl group from the 11 position does not alter the relative orientation of the intercalated chromophore. However, it appears that the phosphorothioate modification influenced the crystallization and caused the 11-deoxydaunomycin-d(CGTsACG) complex to crystallize into a different lattice (space group P2) with different lattice contacts and packing forces than the non-phosphorothioated DNA-anthracycline complexes (space group P4(1)2(1)2). In the minor groove of the DNA, the unexpected position of the amino-sugar of 11-deoxydaunomycin supports the hypothesis that in solution the position of the amino sugar is dynamic.

DNA↗
PubMed2002

Accessibility of phosphates in domain I of 23 S rRNA in the ribosomal 50 S subunit as detected by R(P) phosphorothioates.

Recent atomic models of ribosomal structure emphasize the need for new biochemical methods, suitable for fine-scale studies of ribosomal structure and function. We have used the phosphorothioate approach to probe iodine accessibility of 23 S rRNA domain I phosphates inside functional 50 S ribosomal subunits. Five percent of R(P) isomers of nucleoside phosphorothioate were incorporated into Thermus aquaticus 23 S rRNA during in vitro transcription. Ribosomal large subunits were reconstituted from 23 S rRNA and 5 S rRNA transcripts and ribosomal large subunit proteins. The resulting particles sedimented as 50 S and were active in a peptide bond formation assay. Iodine-induced cleavage sites were determined for domain I of 23 S rRNA by reverse transcriptase-directed primer extension. Specific signals were detected at 360 positions, 80 of which were protected in reconstituted 50 S subunits. We argue that most observed protections are caused by shielding of phosphates by ribosomal proteins. The phosphorothioate approach can be extended to analyze dynamic structural changes during translation and the functional roles of individual chemical groups in rRNA.

Models, Molecular↗
PubMed1998

Solid-phase stereoselective synthesis of 2'-O-methyl-oligoribonucleoside phosphorothioates using nucleoside bicyclic oxazaphospholidines.

The use of 2'-OMe-ribonucleoside bicyclic oxazaphospholidines derived from (R)- or (S)-2-pyrrolidinemethanol has enabled the stereoselective synthesis of (Rp)-, and (Sp)-2'-O-methyloligoribonucleoside phosphorothioates. Interestingly, higher stereoselectivity (96-98%) was observed in the synthesis of (Sp)-2'-O-methyl-oligoribonucleoside phosphorothioates compared to that in the case of (Sp)-oligodeoxyribonucleoside phosphorothioates (90%).

Chromatography, High Pressure Liquid↗
PubMed1998

Mixed-backbone oligonucleotides as second-generation antisense agents with reduced phosphorothioate-related side effects.

Mixed-backbone oligonucleotides containing alternative phosphorothioate and phosphodiester linkages in the 2'-O-methylribonucleosides segment show increased affinity with complementary targets, increased stability towards nucleases in vitro and in vivo, and reduced phosphorothioate-related prolongation of partial thromboplastin time compared to phosphorothioate oligodeoxynucleotides, thereby providing antisense agents with reduced side effects.

Drug Stability↗
PubMed1999

Site of chemical modifications in CpG containing phosphorothioate oligodeoxynucleotide modulates its immunostimulatory activity.

Phosphorothioate oligodeoxynucleotides containing CpG motifs have immunostimulatory activity. Appropriate substitution of deoxynucleosides in the flanking region of CpG-containing phosphorothioate oligodeoxynucleotides with 2'-O-methylribonucleosides results in significant decreases or increases in their immunostimulatory activities. The results provide insights in how to chemically modify phosphorothioate oligodeoxynucleotides containing CpG motifs to suppress or enhance their immunostimulatory activity for different therapeutic uses.

Adjuvants, Immunologic↗
PubMed1996

Nonenzymatic sequence-specific cleavage of duplex DNA via triple-helix formation by homopyrimidine phosphorothioate oligonucleotides.

Phenanthroline was attached covalently to the 5'-terminus of the unmodified and modified (3'-terminal phosphorothioate) oligonucleotide sequences, TTTTTTCTTCTCTTTCC (OP-17 mer) and TTTTTTTCTTCTCTTTCsC (OPRp-17 mer or OPSp-17 mer) via a phosphoramidite bond. Simian virus 40 DNA contains a single target site for these oligonucleotides. In the presence of copper ions, the efficient double-stranded cleavage at 37 degrees C and pH 7.0 was observed by agarose gel electrophoresis. The asymmetric distribution of the cleavage sites on the two strands revealed that the cleavage reaction took place in the minor groove, even though the linker was located in the major groove. Of particular interest are the 3'-terminal phosphorothioate oligonucleotide-phenanthroline derivatives (Rp or Sp), which were found to have cleavage activities of the same order as for the oligonucleotide phenanthroline (OP-17 mer). Furthermore, the OPSp-17 mer was intact after incubation in 10% fetal bovine serum for 24 h, whereas, the OPRp-17 mer was slightly more unstable than the OPSp-17 mer. However, the OP-17 mer was completely degraded. An increased resistance to nucleases has been observed by the introduction of phosphorothioate groups on the 3'-terminus of oligonucleotide-phenanthroline derivatives. This stabilization should help us to design much more efficient chemical recognition enzymes and antisense nucleic acid based anti-viral therapies, which could be used as tools in cellular biology.

Animals↗
PubMed1997

Application of a 5'-bridging phosphorothioate to probe divalent metal and hammerhead ribozyme mediated RNA cleavage.

This paper describes the preparation and application of a chimeric DNA/RNA oligonucleotide that contains a single 5'-bridging phosphorothioate linkage adjacent to a ribonucleotide and embedded in an otherwise all-DNA sequence. The influence of pH, divalent metal cation, hybridization, and secondary structure on the susceptibility of the thio linkage towards transesterification is investigated in an effort to better understand the metal-phosphorothioate interactions and the basis for catalysis. In addition to the chemical cleavage, we have examined the hammerhead ribozyme mediated cleavage of the 5'-bridging phosphorothioate linkage specifically to test the hypothesis that the ribozyme employs a second metal cofactor, which functions as a Lewis acid, to catalyze transesterification. The results of our kinetics experiments do not support this double-metal model.

Base Sequence↗
PubMed2002

Specific phosphorothioate substitutions probe the active site of Bacillus subtilis ribonuclease P.

Ribonuclease P (RNase P) is a ribonucleoprotein that requires magnesium ions to catalyze the 5' maturation of transfer RNA. To identify interactions essential for catalysis, the properties of RNase P containing single sulfur substitutions for nonbridging phosphodiester oxygens in helix P4 of Bacillus subtilis RNase P were analyzed using transient kinetic experiments. Sulfur substitution at the nonbridging oxygens of the phosphodiester bond of nucleotide U51 only modestly affects catalysis. However, phosphorothioate substitutions at A49 and G50 decrease the cleavage rate constant enormously (300-4,000-fold for P RNA and 500-15,000-fold for RNase P holoenzyme) in magnesium without affecting the affinity of pre-tRNA(Asp), highlighting the importance of this region for catalysis. Furthermore, addition of manganese enhances pre-tRNA cleavage catalyzed by B. subtilis RNase P RNA containing an Sp phosphorothioate modification at A49, as observed for Escherichia coli P RNA [Christian et al., RNA, 2000, 6:511-519], suggesting that an essential metal ion may be coordinated at this site. In contrast, no manganese rescue is observed for the A49 Sp phosphorothioate modification in RNase P holoenzyme. These differential manganese rescue effects, along with affinity cleavage, suggest that the protein component may interact with a metal ion bound near A49 in helix P4 of P RNA.

Bacillus subtilis↗
PubMed1998

Structure of 5S rRNA within the Escherichia coli ribosome: iodine-induced cleavage patterns of phosphorothioate derivatives.

The protection patterns of 5S rRNA in solution, within the ribosomal 50S subunit, 70S ribosomes, and functional complexes, were assessed with the phosphorothioate method. About 20% of the analyzed positions (G9-G107) showed strong assembly defects: A phosphorothioate at one of these positions significantly impaired the incorporation of 5S rRNA into 50S particles. The reverse has also been observed: A phosphorothioate is preferred over a phosphate residue in the assembly process at a few positions. The results further demonstrate that 5S rRNA undergoes conformational changes during the assembly in the central protuberance of the 50S subunit and upon association with the small ribosomal subunit forming a 70S ribosome. In striking contrast, when the 70S ribosomes are once formed, the contact pattern of the 5S rRNA is the same in various functional states such as initiation-like complexes and pre- and posttranslocational states.

Base Sequence↗
PubMed1995

The nature of the transition state for enzyme-catalyzed phosphoryl transfer. Hydrolysis of O-aryl phosphorothioates by alkaline phosphatase.

There has been much speculation that enzymes change the nature of the transition state for phosphoryl transfer from the dissociative transition state observed in solution reactions to an associative transition state at the enzyme's active site. This proposal can be tested by comparing linear free energy relationships (LFERs) for nonenzymatic and enzymatic reactions, provided that the specificity of the enzyme's binding site does not perturb the dependence of rate on the intrinsic reactivity of a series of substrates. The shallow binding groove of Escherichia coli alkaline phosphatase (AP) and its wide specificity suggest that this enzyme may be suited for such an approach. A second requirement of this approach is that the actual chemical step is rate-limiting. Comparisons of the reactions of aryl phosphorothioates and aryl phosphates support the previous conclusion that a nonchemical step limits kcat/KM for reactions of aryl phosphates, but suggest that the chemical cleavage step is rate-limiting for the aryl phosphorothioates. We therefore determined the dependence of the rate of AP-catalyzed cleavage of a series of aryl phosphorothioates on the intrinsic reactivity of the substrates. The large negative values of beta leaving group = -0.8 for the enzymatic reaction (kcat/KM) and -1.1 for the nonenzymatic hydrolysis reaction suggest that there is considerable dissociative character in both the enzymatic and nonenzymatic transition states. Despite the wide specificity of AP, certain substrates deviate from the LFER, underscoring that extreme care is required in applying LFERs to enzymatic reactions. The large negative value of beta leaving group suggests that AP can achieve substantial catalysis via a transition state with dissociative character.

Alkaline Phosphatase↗
PubMed2000

Cellular proteins prevent antisense phosphorothioate oligonucleotide (SdT18) to target sense RNA (rA18): development of a new in vitro assay.

There are numerous indications that the "antisense" mechanism alone cannot account for the observed effects in living cells. Despite that, interactions between antisense oligonucleotides (ASO) and cellular proteins are usually not considered. In this work, we have tested the ability of antisense phosphorothioate (SdT) oligonucleotides and natural deoxyoligonucleotides (dT) for their ability to interact with target RNA in the presence of cellular proteins. We show that the affinity for cellular proteins is an essential factor that determines the success of RNA targeting. We have used a simple nuclease digestion assay to detect RNA/ASO hybrid formation in the presence of proteins. The results show the inability of a phosphorothioate oligonucleotide (SdT18) to reach the target RNA (rA18) in vitro in the presence of proteins. However, if proteins are absent, the RNA targeting was successful, as is usual in in vitro assays. Note that the target RNA concentration exceeded physiological values by several orders of magnitude while the crude protein extract was 20-fold diluted in the reaction tube. This finding is compatible with the notion that therapeutic properties of phosphorothioates could largely derive from a so-called "aptamer" effect.

Animals↗
PubMed1982

Phosphorothioate substrates for T4 RNA ligase.

Stereochemical details of the T4 RNA ligase reaction mechanism have been delineated by examining the reactivity of phosphorothioate analogues in the adenosine 5'-triphosphate (ATP) dependent and ATP-independent RNA ligase reactions. Only the SP isomer of the diastereomeric dinucleoside thiopyrophosphate, App(s)I, was active as an activated donor substrate in the ATP-independent RNA ligase reaction. The thiophosphodiester linkage in the ligation product, ApApAp(s)I, that is formed by the reaction of App(s)I (SP) with the oligonucleotide acceptor, ApApA, was shown to have the RP configuration. This indicates that phosphodiester bond formation occurs by a direct, nucleophilic displacement of AMP from App(s)I by the 3'-hydroxyl group of ApApA with inversion of configuration at phosphorus. The adenylylated intermediate, App(s)Ap, that is formed from the phosphorothioate donor, p(s)Ap, in the ATP-dependent RNA ligase reaction was shown to have the same stereochemical configuration as is required for the ATP-independent RNA ligase reaction. These results indicate that RNA ligase maintains a preferred chirality at phosphorus through the adenylylation and ligation steps of the reaction mechanism. An unusual result is the accumulation of adenosine cyclic 2',3'-phosphate 5'-phosphorothioate in the ATP-dependent RNA ligase reaction employing the donor p(s)Ap when the acceptor ApApA is present. This observation suggests that there are two distinct but reactive modes for donor molecules.

Adenine Nucleotides↗
PubMed1984

Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site.

The synthesis and characterization of an octanucleotide, d(GGsAATTCC), containing the recognition sequence of the EcoRI restriction endonuclease with a phosphorothioate internucleotidic linkage at the cleavage site are described. Two approaches for the synthesis of the RP and SP diastereomers of this octamer by the phosphite method are presented. The first consists of the addition of sulfur instead of H2O to the phosphite at the appropriate position during chain elongation. This method results in a mixture of diastereomers that can be separated by high-performance liquid chromatography after 5'-terminal phosphorylation. The second uses the presynthesized and diastereomerically pure dinucleoside phosphorothioate d[Gp(S)A] for the addition to the growing oligonucleotide chain as a block. The products are characterized by digestion with nuclease P1, fast atom bombardment mass spectrometry, 31P NMR spectroscopy, and conversion to d(GGAATTCC) by desulfurization with iodine. Only the RP diastereomers of d(GGsAATTCC) and its 5'-phosphorylated derivative are cleaved by EcoRI endonuclease. The rate of hydrolysis is slower than that of the unmodified octamer. The phosphorothioate octamer will be useful for the determination of the stereochemical course of the EcoRI-catalyzed reaction.

Chromatography, High Pressure Liquid↗
PubMed1980

Stereochemical course of a phosphokinase using a chiral [18O]phosphorothioate. Comparison with the transfer of a chiral [16O,17O,18O]phosphoryl group.

Synthetic adenosine 5'-O-[3-18O,3-thio]triphosphate having the R configuration at the gamma-phosphorus has been used as a substrate in the reaction catalyzed by glycerol kinase. The product sn-glycerol 3-[18O]phosphorothioate has been isolated, and the configuration at phosphorus has been determined by ring closure to the two diastereoisomeric cyclic 2,3-phosphorothioates of sn-glycerol and analysis of the 18O content of each diastereoisomer. The structural identity of these diastereoisomers has been determined by correlation with one of the corresponding diastereoisomers of the cyclic 2,3-phosphorothioate of D-glycerate, whose crystal structure is reported here. From these experiments it is evident that glyc:rol kinase catalyzes the transfer of a thiophosphoryl group with inversion of the configuration at phosphorus, in gratifying agreement with the result from the transfer of a chiral [16O,17O,18O]phosphoryl group [Blättler, W. A., & Knowles, J. R. (1979) J. Am. Chem. Soc. 101, 510].

Adenosine Triphosphate↗
PubMed1979

Stereochemistry of the hydrolysis of the endo isomer of uridine 2',3'-cyclic phosphorothioate catalyzed by the nonspecific phosphohydrolase from Enterobacter aerogenes.

The nonspecific phosphohydrolase from Enterobacter aerogenes (ATCC 13048) requires divalent metal ions for activity, since zinc present in the isolated enzyme can be removed by extensive dialysis against 8-hydroxyquinoline-5-sulfonate at pH 7.5 to yield an inactive enzyme which can be reactivated by addition of Zn2+, Cd2+, Co2+, Mn2+, or Ni2+; six ions of either zinc or cadmium can be incorporated into the inactive enzyme, and this incorporation of metal ion can be correlated with the regaining of activity (J. A. Gerlt, R. Dhesi, and H. C. Hemmings, unpublished experiments). The cadmium-reactivated phosphohydrolase catalyzes the hydrolysis of the endo isomer of uridine 2',3'-cyclic phosphorothioate (U greater than pS) to yield uridine 3'-monophosphorothioate as the major product. After enzymatic hydrolysis of the cyclic phosphorothioate in 19.8% H218O and chemical recyclization of the 18O-labeled acyclic phosphorothioates to yield a mixture of the endo and exo isomers of U greater than pS, 18O is found primarily in the exo isomer, as judged by examination of the 145.7-MHz phosphorus-31 nuclear magnetic resonance spectrum of the mixture. This observation indicates that the cadmium phosphosphohydrolase catalyzes hydrolysis of endo-U greater than pS with inversion of configuration, implying that the hydrolysis reaction proceeds by an in-line attack of water on the phosphorus.

Enterobacter↗
PubMed1998

Biochemical characterization of the HIV-1 integrase 3'-processing activity and its inhibition by phosphorothioate oligonucleotides.

To better understand HIV-1 integrase (IN) functions, we determined the kinetic parameters of the 3'-processing reaction. Steady-state kinetic analysis performed using Dixon plots indicated that the concentration of active enzyme was 10-fold lower than that calculated by protein determination. The turnover number was low, suggesting that IN remained bound to DNA after cleavage. The catalytic efficiency increased 10-fold from 30 to 37 degrees C and 2-fold from 37 to 42 degrees C. In enzyme assays carried out at 37 degrees C, both single- and double-stranded phosphorothioate oligos bound to IN with an efficiency comparable to that of the phosphodiester duplex substrate. The competition efficiency of single-stranded oligos was directly related to the sequence length. On the other hand, phosphorothioate duplex U5 LTRs modified in the plus strand were capable of both competing with the substrate and directly inhibiting the 3'-processing activity. These results suggest that, in addition to other modes of action (inhibition of gp120-CD4 interaction and reverse transcriptase), phosphorothioate hetero- and homopolimeric oligos also potently inhibit the IN activity.

HIV Integrase↗
PubMed1998

Antisense binding enhanced by tertiary interactions: binding of phosphorothioate and N3'-->P5' phosphoramidate hexanucleotides to the catalytic core of a group I ribozyme from the mammalian pathogen Pneumocystis carinii.

Pneumocystis carinii is the most common lethal opportunistic pathogen infecting Acquired Immune Deficiency Syndrome (AIDS) patients, and more effective therapeutics for it are needed. P. carinii, but not humans, contain RNA self-splicing group I introns, so these functionally important RNAs are potential anti-fungal targets. In vitro, d(ATGACT), which mimics the 3' end of the 5' exon of a conserved ribosomal RNA group I intron from mouse-derived Pneumocystis carinii binds to a ribozyme that is a truncated form of this intron. The binding is about 30,000 times tighter than expected for simple base-pairing because binding is enhanced by tertiary interactions. Here we report the effects of modifying the phosphodiester backbone of d(ATGACT) with phosphorothioate and of d(ATGAC)rU with N3'-->P5' phosphoramidate linkages. The enhancement of binding by tertiary interactions is not substantially decreased, and in some cases is increased when single Rp and Sp phosphorothioate substitutions are made, although overall binding is weaker by up to 6-fold. A mixture of 5' exon mimic isomers that each contain five phosphorothioate linkages binds to the ribozyme at least 14-fold less tightly than the corresponding phosphodiester mimic. In contrast, the 5' exon mimic with five internal N3'-->P5' phosphoramidate linkages binds 4-fold more tightly than d(ATGAC)rU. This increased binding is largely due to more favorable base-pairing, but tertiary interactions still enhance binding by more than 2, 000-fold. These results indicate that chemically modified, nuclease stable 5' exon mimics can act as antisense agents with binding enhanced by tertiary interactions (BETI). This strategy permits design of short antisense agents with high specificity.

Animals↗