Synthesis of backbone-modified DNA analogues for biological applications

The phosphite triester method was adapted for automated synthesis of phosphate (backbone)-modified analogues of DNA. The use of phosphoramidite reagents to achieve substitution of the nonequivalent (diastereotopic) oxygens of an internucleotide phosphate linkage is nonselective, and leads to nonequivalent (diastereomeric) strands of DNA with R _p and S _p absolute configurations at the chiral, modified phosphorus position. Indications of the scope and limitations of the use of reversed-phase HPLC to separate these diastereomers have been obtained through studies of backbone-modified DNA analogues having phosphorothioate (P-S), phosphotriester (P-OR), and alkanephosphonate (P-R) linkages. Incorporation of these modifications in the self-complementary octanucleotide d(GGAATTCC) led to separable diastereomers, which form nonequivalent R _p · R _p and S _p · S _p duplexes. A combination of chemical and nuclear Overhauser effect NMR spectroscopic methods was developed to assign unambiguously, for the first time, the absolute configurations at phosphorus in these prototypal cases. The effects of backbone ethylation on DNA structure and dynamics were evaluated by NMR methods. Modified duplexes were used to probe for proposed phosphate contacts for EcoRI endonuclease, and to define, in concert with base-modified analogues, a recognition site for monoclonal anti-native DNA autoantibody.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Preparation of DNA-duplexes, containing internucleotide thiophosphate groups in various positions of the recognition site for the EcoRII restriction endonuclease

Twenty-four 12-mer DNA duplexes, each containing a chiral phosphorothioate group successively replacing one of the internucleotide phosphate groups either in the EcoRII recognition site (5'CCA/TGG) or near to it, were obtained for studying the interaction of the restriction endonuclease EcoRII with internucleotide DNA phosphates. Twelve of the 12-mer oligonucleotides were synthesized as Rp and Sp diastereomeric mixtures. Six of them were separated by reversed-phase HPLC using various buffers. Homogeneous diastereomers of the other oligonucleotides were obtained by enzymatic ligation of the Rp and Sp diastereomers of 5- to 7-mer oligonucleotides preliminarily separated by HPLC with the corresponding short oligonucleotides on a complementary DNA template. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Probing of contacts between <Emphasis Type="Italic">Eco</Emphasis>RII DNA methyltransferase and DNA with the use of substrate analogs and molecular modeling

The molecular mechanisms of DNA recognition and modification by EcoRII DNA methyltransferase (M.EcoRII) were studied using 14-mer substrate analogs containing 2-aminopurine or 1′,2′-dideoxy-D-ribofuranose in the M.EcoRII recognition site. The efficiency of DNA binding and methylation depended on the position of a modified nucleoside residue in the recognition site. A structural model of M.EcoRII in complex with substrate DNA and the cofactor analog S-adenosyl-L-homocysteine (AdoHcy) was constructed using the available crystal structures of M.Hha and M.HaeIII and the recent Frankenstein’s monster approach. The amino acid residues interacting with DNA were predicted based on the model. In addition, theoretical and experimental findings made it possible to predict the groups of atoms of the heterocyclic bases of the M.EcoRII recognition site that are presumably involved in the interactions with the enzyme.     

A nucleotide-independent nitroxide probe reports on site-specific stereomeric environment in DNA

In this report, stereospecific structural and dynamic features in DNA are studied using the site-directed spin labeling technique. A stable nitroxide radical, 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a), was attached postsynthetically to phosphorothioates that were chemically introduced, one at a time, at five sites of a DNA duplex. The two phosphorothioate diastereomers (R_p or S_p) were separated, and nitroxide rotational motions were monitored using electron paramagnetic resonance spectroscopy. The resulting spectra vary according to diastereomer identity and location of the labeling site, with R_p-R5a spectra effectively reporting on local DNA structural features and S_p-R5a spectra sensing variations in local DNA motions. This establishes R_p- and S_p-R5a as unique probes for investigating nucleic acids in a site- and stereospecific manner, which may aid studies of stereospecific DNA/protein interactions. In addition, weighted averages of individual R_p and S_p spectra match those of R5a attached to mixed diastereomers. This suggests that R5a linked to mixed diastereomers reports on the composite behaviors of R_p- and S_p-R5a and is useful in initial probing of the DNA local environment. This work advances understanding of R5a/DNA coupling, and is a key step forward in developing a nucleotide-independent spectroscopic probe for studying nucleic acids.     

Recombinant Components of the EcoRII Restriction–Modification System: Restriction Endonuclease Can Interact with DNA · RNA Duplexes

To obtain recombinant restriction endonuclease (R) and methylase (M) of the EcoRII restriction–modification system, bacterial strains overproducing their functional hexahistidine derivatives were constructed. Active full-length R·EcoRII was produced only in cells that also expressed M·EcoRII from a multicopy plasmid. Recombinant R·EcoRII bound with hybrid DNA·RNA duplexes.     

Comparison of the cleavage profiles of oligonucleotide duplexes with or without phosphorothioate linkages by using a chemical nuclease probe

A manganese porphyrin complex, Mn-TMPyP, associated with KHSO₅ is a chemical nuclease able to selectively recognize the minor groove of three consecutive AT base pairs of DNA and to mediate very precise cleavage chemistry at that particular site. This specific recognition and cleavage were used to probe the accessibility of the minor groove of DNA duplexes composed of one phosphodiester strand and one phosphorothioate strand. The cleavage of 5-GCAAAAGC/5-GCTTTTGC duplexes by Mn-TMPyP/KHSO₅ was monitored by HPLC coupled to electrospray mass analysis. Each single strand was synthesized with all-phosphate, all-Rp-phosphorothioate and all-Sp-phosphorothioate internucleotide bonds. We found that the manganese porphyrin was able to recognize its favorite (AT)₃-box binding site within the heteroduplexes, as in the case of natural DNA. Molecular modeling studies on the interactions of the reactive porphyrin manganese-oxo species with both types of duplexes confirmed the experimental data.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

AFFINITY MODIFICATION OF EcoRII DNA METHYLTRANSFERASE BY THE DIALDEHYDE-SUBSTITUTED DNA DUPLEXES: MAPPING THE ENZYME REGION THAT INTERACTS WITH DNA

ABSTRACT

Affinity modification of EcoRII DNA methyltransferase (M·EcoRII) by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine (Crib*) or 1-(β-D-galactopyranosyl)thymine (Tgal*) residues was performed. Cross-linking yields do not change irrespective of whether active Crib* replaces an outer or an inner (target) deoxycytidine within the EcoRII recognition site. Chemical hydrolysis of M·EcoRII in the covalent cross-linked complex with the Tgal*-substituted DNA indicates the region Gly²⁶⁸-Met³⁹¹ of the methylase that is likely to interact with the DNA sugar-phosphate backbone. Both specific and non-specific DNA interact with the same M·EcoRII region. Our results support the theoretically predicted DNA binding region of M·EcoRII.     

The Effect of the Gene Encoding EcoRII DNA Methyltransferase on the Phenotype of Transgenic Tumor Cell Lines of Nicotiana tabacumand the Methylation Level of CpNpG Sequences in Their Genome

Several tumor cell lines were obtained by transforming Nicotiana tabacumplants with the recombinant Ti plasmid comprising the gene encoding EcoRII DNA methyltransferase (M·EcoRII) and subjected to analysis. The transformed lines differed in their morphology, growth dependence on hormones, and nopaline-synthesizing capacity. Southern blot-hybridization showed that the M·EcoRII gene was present in the cells of all transformed lines. However, genome analysis using polymerase chain reaction with the oligonucleotide primers recognizing 5"-ends of the M·EcoRII gene did not exhibit the full-length copies of the gene. Lower methylation of CpNpG sequences characteristic of all transformed cells could result from the disturbance of one of several plant DNA methyltransferase genes following its homologous recombination with the M·EcoRII gene.     

A Molecular Dynamics Computer Simulation Study of Nucleotide Analogues. Comparison of the Hydration Pattern of Dithymidine Phosphate with those of the Dithymidine Methylphosphonate Diastereomers

Abstract

The hydration pattern of thymidyl(3′→5′) thymidine 1 and those of R_p and S_p diastereomers of the corresponding methylphosphonate analogue 2, have been studied using Molecular Dynamics (MD) computer simulation. It was found that the methylphosphonate modification leads to significant changes in the coordination of water molecules around the internucleotidic linkage and these, in turn, affect the hydration pattern of other parts of the molecule. The most notable differences between R_p and S_p diastereomers 2a and 2b were found to occur at the deoxyribose moieties of the nucleosid-5′-yl units.     

Highly sensitive coupled-column high-performance liquid chromatographic method for the separation and quantitation of the diastereomers of leucovorin and 5-methyltetrahydrofolate in serum and urine

A column-switching chiral HPLC assay was developed that allows the separation and quantitation of the diastereomers of leucovorin (LV, 5-formyltetrahydrofolic acid) and its metabolite 5-methyltetrahydrofolate (METHF) in serum and urine by means of fluorescence detection. The analysis procedure consists of an on-line concentration of the folates in the HPLC system which is followed by the elution and separation of folates on an achiral 3-μm Microbore C₁₈ column in (6R,S)-LV and (6R,S)-LV and (6R,S)-METHF are subsequently transferred on-line onto a chiral 7-μm bovine serum albumin column through a Rheodyne valve system and are separated into their distereometers. Time of analysis is 70 min. Detection limit is 5 ng/ml for each diastereometer. The within-day variation ranges between 3.2 and 15.8% in relation to the measured concentration. Between-day variation is 4.4–12.1% for a concentration of 100 ng/ml for each diastereometer. (6R,S)-LV and (6S)-LV pharmacokinetics were assessed by analyzing serum and urine samples of four-healthy volunteers.     

Evaluation of enantioselectivity in lipase-catalyzed acylation of hydroxyalkylphosphine oxides

The lipase-catalyzed optical resolution of 2-, 3-, and 5-hydroxyalkyl phosphorus compounds 1 provided the corresponding optically pure diastereomers in good yields. (S_P, R)- and (R_P, S)-1 were acylated faster than (S_P, S)- and (R_P, R)-1. The stereoselectivity at the phosphorus atom changed with the flexibility of the active sites in the lipases. The stereoselectivity at the phosphorus atom was higher in the reaction of 1a than in the reaction of 1b,c. The reaction rate of -hydroxyalkylphosphine oxide 1c was faster than that of 1a, although less enantioselectivity was observed at the phosphorus atom.     

UV-induced allevation of lambda restriction in Escherichia coli K-12: kinetics of induction and specificity of this SOS function

Summary In UV-irradiated cells of Escherichia coli K-12 a partial release of the restriction of non-modified phage is observed when the cells are recA ⁺ lexA ⁺. We show here that the induction of this restriction allevation (RA) also depends on the recBC enzyme and that the expression of RA requires protein synthesis. Maximum expression was reached within 60 to 90 min after irradiation. Experiments are presented which show that upon UV-irradiation a signal is created which triggers the development of RA when protein synthesis is allowed. This signal decayed with a half-life of only a few minutes in cells treated with chloramphenicol. The decay kinetics were similar in uvr ⁺ and uvrA mutants. RA appeared to be specific for EcoK insofar as no allevation of restriction by EcoRI, EcoRII and EcoP1 occurred. During maximum expression of RA no gross reduction of the activities of the recBC enzyme (exonuclease V) and the restriction endonuclease EcoK was observed and no new DNA modifying activity appeared in the cells. Since, in fully expressed cells, up to 75% of the infecting DNA was converted to acid-soluble material within 20 min after infection we suggest that only a small specific fraction of infections may undergo RA.     

Antiplatelet Activity,P2Y1 and P2Y12 Inhibition,and Metabolism in Plasma of Stereoisomers of Diadenosine 5′,5′″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate

Background

Diadenosine tetraphosphate (Ap₄A), a constituent of platelet dense granules, and its P¹,P⁴-dithio and/or P²,P³-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y₁ and P2Y₁₂. The most active of those analogs, diadenosine 5′,5″″-P¹,P⁴-dithio-P²,P³-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.

Objective

To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y₁ and P2Y₁₂ receptor antagonism, and their metabolism in human plasma.

Methods

We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y₁-mediated changes in cytosolic Ca²⁺, P2Y₁₂-mediated changes in VASP phosphorylation, and metabolism in human plasma.

Results

The inhibition of ADP-induced human platelet aggregation and human platelet P2Y₁₂ receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P¹- and P⁴-phosphorothioate groups, the S_PS_P diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the R_PR_P diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of S_PR_P diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P²,P³-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the S_PS_P and the R_PR_P diastereomers.

Conclusions

The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y₁ and P2Y₁₂ receptors.     

Lack of correlation between binding of Eco RII methyltransferase to DNA duplexes containing mismatches and the promotion of C to T mutations

The cytosine methyltransferases (MTases) M. HhaIand M. HpaII bind substrates in which the target cytosine is replaced by uracil or thymine, i.e. DNA containing a U:G or a T:G mismatch. We have extended this observation to the EcoRII MTase (M. EcoRII) and determined the apparent K_d for binding. Using a genetic assay we have also tested the possibility that MTase binding to U:G mismatches may interfere with repair of the mismatches and promote C:G to T:A mutations. We have compared two mutants of M. EcoRII that are defective for catalysis by the wild-type enzyme for their ability to bind DNA containing U:G or T:G mismatches and for their ability to promote C to T mutations. We find that although all three proteins are able to bind DNAs with mismatches, only the wild-type enzyme promotes C:G to T:A mutations in vivo. Therefore, the ability of M. EcoRII to bind U:G mismatched duplexes is not sufficient for their mutagenic action in cells. Received: 14 November 1996 / Accepted: 17 February 1997     

An investigation into the solution structure of the single-stranded DNA undecamer 5′d AAGTGTGATAT by means of nuclear Overhauser enhancement measurements

A 500-MHz ¹H-NMR study on the single-stranded DNA undecamer (11-mer) 5d AAGTGTGATAT is presented. Using a combination of one-dimensional pre-steady-state nuclear Overhauser enhancement (NOE) measurements and two-dimensional homonuclear J-correlated spectroscopy, virtually complete resonance assignments are obtained. The relative magnitudes of the intra- and internucleotide NOEs indicate that the overall structure of the single-stranded 11-mer is a right-handed B-type helix with extensive base stacking. Within this overall structure there is quite a large degree of variability, as exemplified by variations in glycosidic bond and sugar pucker conformations, most likely determined by base sequence.Abbreviations NOE nuclear Overhauser effect - COSY twodimensional homonuclear J-correlated spectroscopy - 11-mer undecamer - EDTA sodium ethylenediamine tetraacetate - HPLC nigh-pressure liquid chromatography - DSS 4,4-dimethylsilapentane-1-sulfonate     

DNA-binding and molecular mechanics modelling studies of the bulky chiral platinum(II) complex [PtCl2(mepyrr)] (mepyrr = N-methyl-2-aminomethylpyrrolidine)

Detailed studies were carried out on the binding of the enantiomers of [PtCl₂(mepyrr)] (mepyrr = N-methyl-2-aminomethylpyrrolidine) to dG, d(GpG) and a 52-mer oligonucleotide. The pyrrolidine ligand structure was found to be neither sufficiently rigid nor bulky to enforce a single chirality at the exocyclic amine site in this complex, resulting in the presence of diastereomers that complicated the binding studies. Reaction of the (GpG) dinucleotide with R- and S-[PtCl₂(mepyrr)] resulted in formation of four [Pt{d(GpG)}(mepyrr)] isomers for each enantiomer as a consequence of the existence of two orientational isomers and two diastereomers. These isomers formed in different amounts most likely as a consequence of the unequal formation of the diastereomers together with stereoselectivity induced by interactions between the dinucleotide and the mepyrr ligand. The [PtCl₂(mepyrr)] complexes displayed stereoselectivity and enantioselectivity in their reactions with a 52-mer duplex designed to allow formation of only GpG intrastrand adducts. All four bifunctional adducts formed for each enantiomer, providing further evidence of the lack of directing ability of the ligand in formation of the 1,2-intrastrand adduct. Significant amounts of monofunctional species remained in these assays suggesting that the introduction of the methyl substituent to the exocyclic amine inhibited ring-closure to the bifunctional adduct. This was not sufficient to achieve enantiospecificity, but in the case of the R-enantiomer, one of the bifunctional adducts formed in only small amounts.     

Factors encoded by and affecting the holding stability of yeast plasmid pSR1

Summary A 2 m DNA-like plasmid, pSR1, isolated from a strain of Zygosaccharomyces rouxii has three coding frames, P, S and R. Insertional inactivation of R completely abolished the intramolecular recombination, and the defect was complemented by an intact R frame on a coexistent plasmid molecule. The P and S regions were also transactive and important, but not essential, for the stable maintenance of the plasmid molecules. Insertional disruption of the P frame suggested that it produces a protein factor. Similar insertional disruption of the S frame affected the plasmid stability in Z. rouxii and Saccharomyces cerevisiae hosts differently, depending on whether the inserted DNA fragment was a short 8 bp SalI linker or a long (2.2 kb) DNA fragment. Results strongly suggested that the S region encodes two factors, one RNA and the other a protein, and that the S protein is compatible with a sprecific hostfactor in Z. rouxii, but not in S. cerevisiae. In addition, a cis-acting locus, Z, was found at a site in the plasmid molecule where no distinct open reading frames were located. No long direct repeats or inverted repeats were observed in the Z region, such as are found in the REP3 locus of 2 m DNA.     

DNA Duplexes Containing Altered Sugar Residues as Probes of EcoRII and MvaI Endonuclease Interactions with Sugar-Phosphate Backbone

Abstract

Oligonucleotides containing 1-(β-D-2′-deoxy-threo-pentofuranosyl)cytosine (dCx) and/or 1-(β-D-2′-deoxy-threo-pentofuranosyl)thymine (dT_x) in place of dC and dT residues in the EcoRII and MvaI recognition site CC^A/_TGG were synthesized in order to investigate specific recognition of the DNA sugar-phosphate backbone by EcoRII and MvaI restriction endonucleases. In 2′-deoxyxylosyl moieties of dC_x and dT_x, 3′-hydroxyl groups were inverted, which perturbs the related individual phosphates. Introduction of a single 2′-deoxyxylo-syl moiety into a dC·dG pair resulted in a minor destabilization of double-stranded DNA structure. In the case of a dA·dT pair the effect of a 2′-deoxyxylose incorporation was much more pronounced. Multiple dC_x modifications and their combination with dT_x did not enhance the destabilization effect. Hydrolysis of dC_x-containing DNA duplexes by EcoRII endonuclease was blocked and binding affinity was strongly depended on the location of an altered sugar. A DNA duplex containing a dT_x residue was cleaved by the enzyme, but k_cat/K_M was slightly reduced. In contrast, MvaI endonuclease efficiently cleaved both types of sugar-altered substrate analogs. However it did not cleave conformationally perturbed scissile bonds, when the corresponding unmodified bonds were perfectly hydrolyzed in the same DNA duplexes. Based on these data the possible contributions of individual phosphates in the recognition site to substrate recognition and catalysis by EcoRII were proposed. We observed strikingly non-equivalent inputs for different phosphates with respect to their effect on EcoRII-DNA complex formation.     

Molecular cloning of EcoRII endonuclease and methylase genes

Summary The genes for restriction-modification system EcoRII have been cloned from plasmid N3 DNA using RSF2124 as a vector plasmid. The hybrid plasmids designated pFK321 and pFK322 contained a 5.8 megadaltons EcoRI — fragment derived from N3 DNA including the genes for restriction-modification system EcoRII and a gene for resistance to sulfanilamide.     

Purification and properties of ap-nitrobenzyl esterase fromBacillus subtilis

A procedure for purifying to homogeneity a microbially produced biocatalyst useful for deblocking intermediates in the manufacture of beta-lactam antibiotics is reported. In aqueous solution the purifiedp-nitrobenzyl (PNB) carboxy-esterase was soluble, monomeric (molecular weight: 54 000 by SDS-PAGE or by gel filtration) and exhibited an acidic pl, 4.1. The PNB carboxy-esterase catalyzed rapid ester hydrolysis for simple organic esters such as PNB-acetate, benzyl acetate and -naphthyl acetate and catalyzed deblocking (ester hydrolysis) of beta-lactam antibiotic PNB esters such as cephalexin-PNB and loracarbef-PNB. TheN-terminal amino acid sequence and the amino acid composition are reported. A serine residue is involved in ester hydrolysis: the PNB carboxy esterase was inhibited by phenylmethylsulfonyl fluoride and diethylp-nitrophenyl phosphate; one mole of diisopropyl fluorophosphate titration was required per mole of PNB carboxy-esterase for complete inhibition. When the [³H]-diisopropyl fluorophosphate-treated biocatalyst was digested with Lys C and the resulting peptides separated by HPLC, a single [³H]-labeled peptide was obtained; its amino acid sequence is reported. Inhibition of the PNB carboxy esterase by diethyl pyrocarbonate suggests that a histidinyl residue (or residues) is (are) also involved in the catalytic site of the esterase.Abbreviations used -ME -mercaptoethanol - Cf cefaclor - Cf nucleus-PNB - (6R, 7R) 7-amino-3-chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester - Cp cephalexin - Cp-PNB p-nitrobenzyl carboxy-ester of cephalexin - DEPC diethyl, pyrocarbonate - DFP diisopropyl fluorophosphate - DMSO dimethyl sulfoxide - DNP diethylp-nitrophenyl phosphate - EDTA ethylenediaminetetraacetic acid - EGTA ethylene, glycol-bis(aminoethyl ether) - N,N,NN tetracetic acid - Lc loracarbef - Lc-PNB p-nitrobenzyl carboxy-ester of loracarbef - Lc nucleus-PNB - (6R, 7S) 7-amino-3-chloro-8-oxo-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester - Lys C an endoproteinase specifically cleaving at C terminal lysine residues - MW_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - PNB p-nitrobenzyl - PNBCE p-nitrobenzyl carboxy-esterase - SDS sodium dodecyl sulfate