Inhibition of DNA chain elongation by a phosphotriester linkage in template oligodeoxyribonucleotides.

We synthesized two oligodeoxyribonucleotides bearing an isopropyl phosphotriester at defined positions using new phosphorobisamidite chemistry. Diastereomers were separated with HPLC and their template properties were analyzed. Priming oligodeoxyribonucleotides labeled with 32P at the 5'-end were annealed to the modified oligodeoxyribonucleotides and elongated with DNA polymerase I large fragment from Escherichia coli. Results show that the phosphotriester inhibits the DNA chain elongation partially and the extents of the inhibition are remarkably different between the two diastereomers.     

Differences in replication of a DNA template containing an ethyl phosphotriester by T4 DNA polymerase and Escherichia coli DNA polymerase I

A DNA template containing a single ethyl phosphotriester was replicated in vitro by the bacteriophage T4 DNA polymerase and by Escherichia coli DNA polymerase I (DNA pol I). Escherichia coli DNA pol I bypassed the lesion efficiently, but partial inhibition was observed for T4 DNA polymerase. The replication block produced by the ethyl phosphotriester was increased at low dNTP concentrations and for a mutant T4 DNA polymerase with an antimutator phenotype, increased proofreading activity, and reduced ability to bind DNA in the polymerase active center. These observations support a model in which an ethyl phosphotriester impedes primer elongation by T4 DNA polymerase by decreasing formation of the ternary DNA polymerase–DNA–dNTP complex. When primer elongation is not possible, proofreading becomes the favored reaction. Apparent futile cycles of nucleotide incorporation and proofreading, the idling reaction, were observed at the site of the lesion. The replication block was overcome by higher dNTP concentrations. Thus, ethyl phosphotriesters may be tolerated in vivo by the up-regulation of dNTP biosynthesis that occurs during the cellular checkpoint response to blocked DNA replication forks.     

Synthesis of the human insulin gene. Part II. Further improvements in the modified phosphotriester method and the synthesis of seventeen deoxyribooligonucleotide fragments constituting human insulin chains B and mini-CDNA.

The purification of protected deoxyribooligonucleotides containing phosphotriester internucleotidic linkages has been improved by developing a deactivated silica gel chromatographic technique. The efficiency of this technique as applied in the modified phosphotriester approach has been demonstrated in the rapid synthesis of seventeen pure fragments constituting the sequence of human insulin B and mini-C DNA. The sequence of each oligomer was confirmed by the two-dimensional mobility shift method of fingerprinting.     

Synthesis of the human insulin gene. Part III. Chemical synthesis of 5'-phosphomonoester group containing deoxyribooligonucleotides by the modified phosphotriester method. Its application in the synthesis of seventeen fragments constituting human insulin C-chain DNA.

A method for phosphorylating a protected deoxyribooligonucleotide containing phosphotriester linkages is described. The modified phosphotriester method of chemical synthesis is further refined in terms of (i) better final deblocking conditions and (ii) new chromatography solvent systems containing acetone-water-ethyl acetate to yield pure oligomers. The effectiveness of these improvements has been demonstrated in the rapid and efficient synthesis of seventeen fragments constituting the sequence of human insulin C-chain DNA.     

Computational analysis of the effects of site-specific phosphate alkylation in the DNA oligomer (d-[GGAATTCC])2

Alkylation of the sugar-phosphate backbone of DNA can result upon exposure to several potent carcinogens, inducing DNA misfunction. In order to assess the structural and energetic changes in DNA helices induced by such alkylation, we have performed AMBER-based analyses on phosphotriester containing analogues of (d-[GGAATTCC])2. Fourteen analogues of the nonalkylated oligomer were examined, each bearing a single alkylation of known stereochemistry. Results indicate that although there is minimal effect on the aromatic bases, the presence of a phosphotriester disturbs the sugar-phosphate backbone in complex ways. For most analogues, total minimum energies are lower for the Sp-alkylations than for the Rp-alkylations which point directly into the major groove of the helix; however, different energetic contributions follow different, or no, trends in dependence on alkylation site and/or stereochemistry. Where data is available, experimental nmr results agree with the calculations reported here.     

N-azidomethylbenzoyl blocking group in the phosphotriester synthesis of oligonucleotides

An effective modification of phosphotriester method for automatic synthesis of DNA and RNA fragments using O-nucleophilic intramolecular catalysis and 2-(azidometil)benzoyl group to protect amino groups of heterocyclic bases of nucleotides is described.     

<Emphasis Type="Italic">N</Emphasis>-azidomethylbenzoyl blocking group in the phosphotriester synthesis of oligoribonucleotides

An effective modification of the phosphotriester method has been developed for the automatic synthesis of DNA and RNA fragments using O-nucleophilic intramolecular catalysis and the 2-(azidomethyl)benzoyl group for the protection of the amino groups of nucleotide heterocyclic bases.     

Mutagenesis directed by phosphotriester analogs of oligonucleotides: a way to site-specific mutagenesis in vivo

A new approach is proposed to obtain the directed mutations in the gene under study. The technique is based on using alkylphosphotriester analogues of oligodeoxyribonucleotides as site-specific mutagens. The deletion C in lacZ' gene of bacteriophage M13mpB was obtained by cotransfection of Escherichia coli cells with a mix of DNA and phosphotriester analogues of oligonucleotides.     

Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages

Several classes of oligonucleotide antisense compounds of sequence complementary to the start of the mRNA coding sequence for chloramphenicol acetyl transferase (CAT), including methylphosphonate, alkyltriester, and phosphorothioate analogues of DNA, have been compared to "normal" phosphodiester oligonucleotides for their ability to inhibit expression of plasmid-directed CAT gene activity in CV-1 cells. CAT gene expression was inhibited when transfection with plasmid DNA containing the gene for CAT coupled to simian virus 40 regulatory sequences (pSV2CAT) or the human immunodeficiency virus enhancer (pHIVCAT) was carried out in the presence of 30 microM concentrations of analogue. For the oligo-methylphosphonate analogue, inhibition was dependent on both oligomer concentration and chain length. Analogues with phosphodiester linkages that alternated with either methylphosphonate, ethyl phosphotriester, or isopropyl phosphotriester linkages were less effective inhibitors, in that order. The phosphorothioate analogue was about two-times more potent than the oligo-methylphosphonate, which was in turn approximately twice as potent as the normal oligonucleotide.     

Synthesis of Oligonucleotides Containing the Abasic Site Model Compound 1,4-Anhydro-2-Deoxy-D-Ribitol

Abstract

An improved approach for the synthesis of phosphotriester and phosphoramidite derivatives of the 1,4-anhydro-2-deoxy-D-ribitol is presented. The incorporation of these compounds in synthetic DNA and the insertion of purine deoxyribonucleotides opposite the reduced abasic site by DNA polymerase is described.     

Template properties of DNA alkylated with N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea

Alkylation of DNA with N-methyl-N-nitrosourea (MeNU) and N-ethyl-N-nitrosourea (EtNU) reduces its ability to support RNA synthesis catalyzed by exogenously added RNA polymerase. It is likely that 7-alkylguanine and alkyl phosphotriester in DNA are mainly responsible for the inhibition of RNA synthesis. The inhibitory effect of alkyl groups varies depending upon divalent metal ions and the type of RNA polymerase used as well as upon the presence of chromosomal proteins on DNA templates. Analyses of RNA products indicate that inhibition occurs primarily at the initiation step.     

Comparison of controlled pore glass and Kieselguhr-polydimethylacrylamide composite as supports for solid-phase synthesis of 23-residue oligodeoxyribonucleotides in milligram amounts

Two 23-residue oligodeoxyribonucleotides, corresponding to both strands of a DNA duplex at the OR3 site of bacteriophage lambda, have been synthesized in good yields and in milligram quantities by a solid-phase phosphotriester method using two different supports, Kieselguhr-polydimethylacrylamide composite and controlled pore glass. Rapid purification was possible using high-performance liquid chromatography on radial compression ion-exchange columns. The results and utility of the two supports are compared.     

A general method for the synthesis of 5'-monophosphates of DNA fragments via phosphotriester intermediates.

A new and attractive phosphorylation procedure which allows the introduction, via phosphotriester intermediates, of 5'-phosphate functions of DNA fragments is described. The method is based on the activation of bifunctional phosphorylating agents with 1-hydroxybenzotriazole. The approach will be exemplified by the synthesis of pACGC using four different 5'-phosphotriester intermediates.     

Mutagenesis, induced by phosphotriester analogs of oligonucleotides and directed to the cleavage site of double-spiral DNA]

The mutagenic properties of phosphotriester analogues revealed in course of interaction with linearized plasmid DNA were studied. The plasmid-based model system permitting one to test reliably the induced mutations is proposed. The efficiency of mutagenesis was shown to depend on the length of the oligonucleotide-mutagen and the genotype of the transformed Escherichia coli strain. The possible mechanisms involved in mutagenesis are discussed.     

Chemico-enzymatic synthesis of an oligonucleotide template for an analog of ACTH (4-10) fragment

Six oligodeoxyribonucleotides ranging from 9-mer to 13-mer were synthesized in solution by the phosphotriester approach and enzymatically joined by T4 DNA ligase. The obtained double-stranded DNA (32 b.p.) with protruding 5'-ends corresponding to the recognition sites for restrictases EcoRI and BamHI represents an oligonucleotide template coding for the modified amino acid sequence 4-10 of the adrenocorticotropic hormone, [Pro8,Gly9,Pro10]ACTH-(4-10).     

E. coli Ada regulatory protein repairs the SP diastereoisomer of alkylated DNA

Using HPLC and 31P NMR spectroscopy on a chemically synthesized asymmetric mixture of the diastereoisomers of thymidyl(3'----5')thymidyl-O-methyl phosphate absolute configuration has been correlated with chromatographic mobility. The methyl phosphotriester system in alkylated DNA which is repaired by the Ada regulatory protein of E. coli has consequently been established to possess the Sp configuration.     

New effective method for the synthesis of oligonucleotides via phosphotriester intermediates.

A rapid and convenient method for the synthesis of deoxyribooligonucleotides has been developed using the phosphotriester approach. The advantage of this methodology for work in solution was successfully demonstrated in synthesis of a number of DNA fragments up to 32-long. Adaptation of the presented method to solid-phase synthesis allows a pentadecamer to be assembled in 4-5 hours using dinucleotides as coupling units.     

Mutant Escherichia coli Ada proteins simultaneously defective in the repair of O6-methylguanine and in gene activation.

The activated Ada protein triggers expression of DNA repair genes in Escherichia coli in response to alkylation damage. Ada also possesses two distinct suicide alkyltransferase activities, for O6-alkylguanines and for alkyl phosphotriesters in DNA. The mutant Ada3 and Ada5 transferases repair O6-methylguanine in DNA 20 and 3000 times more slowly, respectively, than the wild-type Ada protein, but both exhibit normal DNA phosphotriester repair. These same proteins also exhibit delayed and sluggish induction of the ada and alkA genes. Since the C-terminal O6-methylguanine methyltransferase domain of Ada is not implicated in the direct binding of specific DNA sequences, this part of the Ada protein is likely to play an alternative mechanistic role in gene activation, either by promoting Ada dimerization, or via direct contacts with RNA polymerase.     

Modulation of binding properties of amphiphilic DNA containing multiple dodecyl phosphotriester linkages to lipid bilayer membrane

DNA is a promising functional molecule to modify and design lipid membrane functions. In order to use DNA in a hydrophilic–hydrophobic interface including lipid membrane, we have developed an amphiphilic DNA having dodecyl phosphotriester linkages (dod-DNA). Herein, we report the binding of a series of amphiphilic dod-DNAs to the lipid bilayer membrane. Surface plasmon resonance (SPR) assay and fluorescent microscopy showed that dod-DNA having three dodecyl groups at each end strongly bound to lipid membrane due to the slow dissociation rate and the dod-DNA can be used as a linear template for molecular arrangement on the membrane surface.