Isolation of a specific chromosomic DNA sequence of Bacillus anthracis and its possible use in diagnosis

Abstract A 277-bp long DNA fragment, Ba813, was isolated from an avirulent Bacillus anthracis strain 7700 genomic library. Two oligonucleotides derived from the Ba813 sequence were used as primers in polymerase chain reaction tests on genomic DNA from 28 Bacillus anthracis and from 33 heterologous bacteria strains. A specific, 152-bp long DNA fragment was amplified only when Bacillus anthracis DNA was used as the target. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide C1 was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide D3 was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. Amplification of Ba813 sequence may provide the basis for rapid and reliable assay for the detection and identification of Bacillus anthracis .     

Synthesis and properties of triple helix-forming oligodeoxyribonucleotides containing 7-chloro-7-deaza-2'-deoxyguanosine.

Multiple incorporations of 7-chloro-7-deaza-2'-deoxyguanosine in place of 2'-deoxyguanosine have been performed into a triple helix-forming oligodeoxyribonucleotide involving a run of six contiguous guanines designed to bind in a parallel orientation relative to the purine strand of the DNA target. The ability of these modified oligodeoxyribonucleotides to form triple helices in a buffer containing monovalent cations was studied by UV--melting curves analysis, gel shift assay and restriction enzyme protection assay. In the presence of Na(+), the incorporation of two, three or five modified nucleosides in the third strand has improved the efficacy of formation of the triplex as compared to that formed with the unmodified oligonucleotide. The stabilities of the three modified triplexes were similar. The coupling of 6-chloro-2-methoxy-9-(omega-hexylamino)-acridine to the 5'-end of the oligonucleotides containing modified nucleosides led to an increase in triplex stability similar to that observed when the acridine was added to the 5'-end of the unmodified oligonucleotide. In the presence of K(+), only the oligodeoxyribonucleotides containing modified G retained the ability to form triple helices with the same efficiency. The incorporation of the modified nucleoside has two effects: (i) it decreases TFO self-association, and (ii) it slightly increases triplex stability. The enhanced ability of the modified oligonucleotides containing 7-chloro-7-deaza-2'-deoxyguanosine over the parent oligomer to form triple helices was confirmed by inhibition of restriction enzyme cleavage using a circular plasmid containing the target sequence.     

Inhibition of dengue virus by novel, modified antisense oligonucleotides.

Five different target regions along the length of the dengue virus type 2 genome were compared for inhibition of the virus following intracellular injection of the cognate antisense oligonucleotides and their analogs. Unmodified phosphodiester oligonucleotides as well as the corresponding phosphorothioate oligonucleotides were ineffective in bringing about a significant inhibition of the virus. Novel modified phosphorothioate oligonucleotides in which the C-5 atoms of uridines and cytidines were replaced by propynyl groups caused a significant inhibition of the virus. Antisense oligonucleotide directed against the target region near the translation initiation site of dengue virus RNA was the most effective, followed by antisense oligonucleotide directed against a target in the 3' untranslated region of the virus RNA. It is suggested that the inhibitory effect of these novel modified oligonucleotides is due to their increased affinity for the target sequences and that they probably function via an RNase H cleavage of the oligonucleotide:RNA heteroduplex.     

Theoretical analysis of ''addressed'' chemical modification of DNA.

Chemical "addressed" modification of DNA involves treatment of single-stranded DNA with oligonucleotides complementary to certain target sequences in this DNA and bearing a groupings reactive towards DNA bases. The binding of oligonucleotides can occur both at completely (specific) and incompletely (nonspecific) complementary sites. We analyse the modification of a fragment that is flanked by two target sequences complementary to a given oligonucleotide address, contains no more such targets and has some randomly distributed sites for nonspecific binding. Conditions for the maximum ratio between specific and non-specific modification are determined. We find the probability of both target termini being specifically modified without any non-specific modification occurring within the fragment up to a given moment in time. Quantitative analysis is based on the use of known features of the specific and non-specific binding of an oligonucleotide to DNA sites. This analysis shows the possibility of specific cutting of DNA based on addressed modification.     

Complementary addressed modification and cleavage of a single stranded DNA fragment with alkylating oligonucleotide derivatives.

A single stranded DNA fragment was modified with alkylating derivatives of oligonucleotides complementary to a certain nucleotide sequences in the fragment. The derivatives carried aromatic 2-chloroethylamino groups at their 3'- or 5'-terminal nucleotide residues. Some of the derivatives carried both alkylating group and intercalating phenazine group which stabilized complementary complexes. It was found that these oligonucleotide derivatives modify the DNA fragment in a specific way near the target complementary nucleotide sequences, and the DNA fragment can be cleaved at the alkylated nucleotides positions. Alkylating derivatives carrying phenazine groups were found to be the most efficient in reaction with the DNA fragment.     

Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation.

As an alternative to standard gene transfer techniques for genetic manipulation, we have investigated the use of triple helix-forming oligonucleotides to target mutations to selected genes within mammalian cells. By treating monkey COS cells with oligonucleotides linked to psoralen, we have generated targeted mutations in a simian virus 40 (SV40) vector contained within the cells via intracellular triple helix formation. Oligonucleotide entry into the cells and sequence-specific triplex formation within the SV40 DNA deliver the psoralen to the targeted site. Photoactivation of the psoralen by long-wavelength UV light yields adducts and thereby mutations at that site. We engineered into the SV40 vector novel supF mutation reporter genes containing modified polypurine sites amenable to triplex formation. By comparing the abilities of a series of oligonucleotides to target these new sites, we show that targeted mutagenesis in vivo depends on the strength and specificity of the third-strand binding. Oligonucleotides with weak target site binding affinity or with only partial target site homology were ineffective at inducing mutations in the SV40 vectors within the COS cells. We also show that the targeted mutagenesis is dependent on the oligonucleotide concentration and is influenced by the timing of the oligonucleotide treatment and of the UV irradiation of the cells. Frequencies of intracellular targeted mutagenesis in the range of 1 to 2% were observed, depending upon the conditions of the experiment. DNA sequence analysis revealed that most of the mutations were T.A-to-A.T transversions precisely at the targeted psoralen intercalation site. Several deletions encompassing that site were also seen. The ability to target mutations to selected sites within mammalian cells by using modified triplex-forming oligonucleotides may provide a new research tool and may eventually lead to therapeutic applications.     

Pyrimidine morpholino oligonucleotides form a stable triple helix in the absence of magnesium ions

Oligonucleotides can be used as sequence-specific DNA ligands by forming a local triple helix. In order to form more stable triple-helical structures or prevent their degradation in cells, oligonucleotide analogues that are modified at either the backbone or base level are routinely used. Morpholino oligonucleotides appeared recently as a promising modification for antisense applications. We report here a study that indicates the possibility of a triple helix formation with a morpholino pyrimidine TFO and its comparison with a phosphodiester and a phosphoramidate oligonucleotide. At a neutral pH and in the presence of a high magnesium ion concentration (10 mM), the phosphoramidate oligomer forms the most stable triple helix, whereas in the absence of magnesium ion but at a physiological monovalent cation concentration (0.14 M) only morpholino oligonucleotides form a stable triplex. To our knowledge, this is the first report of a stable triple helix in the pyrimidine motif formed by a noncharged oligonucleotide third strand (the morpholino oligonucleotide) and a DNA duplex. We show here that the structure formed with the morpholino oligomer is a bona fide triple helix and it is destabilized by high concentrations of potassium ions or divalent cations (Mg(2+)).     

Repair of triple helix directed psoralen adducts in human cells.

Triple helix forming oligonucleotides can direct DNA damaging agents at specific sites in an intact double helix. In our study, triple helix formation was demonstrated in a SV40 based shuttle vector treated with psoralen linked to a 22-mer purine rich oligonucleotide. UVA irradiation caused a covalent linkage of the oligonucleotide through the psoralen to the mutational supF marker gene of the plasmid. After passage in the Jurkat human cell line the recovered vector was analysed in an indicator bacterial strain and mutants were collected. The presence of adducts in the target sequence did not reduce the yield of replicated progeny vector molecules, indicating repair of triple helix associated monoadducts and cross-links. Mutations were highly targeted to a six nucleotide long region of the target sequence. The number of target sequence mutants obtained after triple helix directed psoralen treatment was approximately 160 times higher than with free psoralen. A further investigation of the exact mechanism of the mutational process could make triple helix directed mutagenesis a more useful tool in gene therapy, antiviral therapy, and in studies on DNA repair and genome organisation.     

Biosensor detection of triplex formation by modified oligonucleotides

Due to the instability of DNA oligonucleotides in biological solutions, antisense or antigene therapies aimed at modulation of specific gene expression will most likely require the use of oligonucleotides with modified backbones. Here, we examine the use of a surface plasmon resonance biosensor (BIAcore) to compare triplex-directed binding of modified oligonucleotides targeted to a region of the murine c-myc promoter. We describe optimization of experimental conditions to minimize nonspecific interactions between the oligonucleotides and the sensor chip surface, and the limitations imposed by certain backbones and sequence types. The abilities of pyrimidine oligonucleotides with various modified backbones to form specific triple helices with an immobilized hairpin duplex were readily determined using the biosensor. Modification of the third-strand oligonucleotide with RNA or 2(')-O-methyl RNA was found to enhance triplex formation, whereas phosphorothioate or phosphotriester substitutions abrogated it. A comparison of these results to DNase I footprinting experiments using the same oligonucleotides showed complete agreement between the two sets of data.     

Roles of the TGACT repeat sequence in the yeast TRP5 promoter

Yeast genes under general amino acid control contain multiple copies of a sequence known as the TGACT repeat in the 5'-flanking DNA. The yeast TRP5 gene contains two copies of the TGACT repeat sequence in its 5'-flanking region. The upstream TGACT repeat of TRP5 is required for normal basal expression as well as derepression by general control. Synthetic oligonucleotides containing a TGACT sequence were inserted into previously constructed TRP5 control region deletion mutants. A synthetic 17-base pairs (bp) oligonucleotide containing a TGACT copy along with flanking nucleotides from HIS3 was able to restore derepression in all deletion mutants tested. The 17-bp oligonucleotide also functioned bidirectionally. Replacements in the upstream control region by synthetic oligonucleotides indicated that sequences other than the TGACT repeat are required for high basal expression. Replacements of the downstream repeat sequence by the 17-bp oligonucleotide suggest its main role in this position is for derepressed expression. High level derepressed expression was found to correlate with the presence of two repeats.     

Rapid detection of Salmonella subspecies I by PCR combined with non-radioactive hybridisation using covalently immobilised oligonucleotide on a microplate

Abstract A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella . One pair of oligonucleotide primers was designed to amplify a 93-bp fragment of a gene required for the invasion of HeLa cells by Salmonella ser Typhi strain Ty2. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. The described combination of microplate sandwich hybridisation and PCR seems to be a suitable method for rapid detection of Salmonella subspecies I. It only requires a thermal cycler and a conventional microtiter reader, and can be readily done on a large scale.     

Chemical modification of pyrimidine TFOs: effect on i-motif and triple helix formation

In order to form more stable triple helical structures or to prevent their degradation in cells, oligonucleotide analogs are routinely used, either in the backbone or among the bases. The target sequence chosen for this study is a 16-base-long oligopurine-oligopyrimidine region present in the human neurotrophin 4/5 gene. Seven different chemical modifications were tested for their effect on (i) triple helix formation and (ii) i-DNA stability. i-DNA is a tetrameric structure involving hemiprotonated C x C+ base pairs, which may act as a competing structure for triplex formation, especially in the case of a cytosine-rich third strand. At acid pH, oligophosphoramidates formed the most stable triple helix, whereas oligonucleotides including 5-propynyl-dU formed a stable i-motif which precluded triplex formation. Only two candidates stabilized triple helices at neutral pH: oligonucleotides with phosphoramidate linkage and phosphodiester oligonucleotides containing 5-methyl-dC and 5-propynyl-dU.     

Characterization of the aac(6'')-Ik gene of Acinetobacter sp. 6

Abstract Chromosomal DNA of different species of mycobacteria, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium avium and Mycobacterium smegmatis , has been submitted to polymerase chain reaction using two oligonucleotide primers highly homologous to DNA sequences flanking the quinolone resistance-determining region in the gyrA gene of Escherichia coli and Staphylococcus aureus . For each of these mycobacterial species, a 150-bp DNA fragment hybridizing with an intragenic probe of the gyrA gene of E. coli K12 was obtained. The nucleotide sequences of the 108-bp fragments amplified from M. tuberculosis and M. avium were determined. The two sequences were 87% homologous. Except for one residue, their deduced amino acid sequences were identical and shared 67% homology with the quinolone resistance-determining region of the gyrase A subunits of E. coli and S. aureus . Sequencing of the 108-bp fragment amplified from an in vitro mutant of M. avium , highly resistant to fluoroquinolones, showed a point mutation leading to the substitution of Ala for Val at a position corresponding to residues involved in quinolone resistance in E. coli and S. aureus , i.e. Ser 83 for E. coli and Ser 84 for S. aureus .     

Formation of nucleosomes does not suppress interaction of a DNA fragment with an alkylating derivative of a pyrimidine oligonucleotide

Oligonucleotide derivatives capable of binding to specific nucleic acids are considered as potential therapeutic agents, exerting their action at the level of genome functioning (Hélène, 1991; Knorre et al., 1993). A straightforward approach to targeting DNA is based on using oligonucleotides capable of binding to oligopurine-oligopyrimidine sequences by formation of triple-strand structures. We report results of experiments on sequence-specific chemical modification of a 490-bp fragment of pfosCAT plasmid, containing the promoter segment of the c-fos gene using 4-(N-2-chloroethyl-N-methylamino)-benzylphosphamide derivatives of a homopyrimidine 14-mer oligonucleotide. It was shown that in both the free DNA and the DNA involved in nucleosome structure, reaction occurred with similar efficiency at the target guanosine residue G404.     

Application of polymerase chain reaction with specific and arbitrary primers to identification and differentiation of Leishmania parasites

Abstract Two oligonucleotide primers Lsmc1 and Lsmv1 derived from the conserved and the variable region of a major class kinetoplast DNA (kDNA) minicircle (pLURkE3) of Leishmania strain UR6 were used for the polymerase chain reaction (PCR) in order to amplify a 461-bp fragment from the kDNAs of different Leishmania species. These primers amplify the specific fragment from the kDNAs of cutaneous species only. The cutaneous species can further be distinguished by randomly amplified polymorphic DNA (RAPD) analysis of the kDNAs of these organisms using arbitrarily chosen oligonucleotides. The arbitrary primers also generate polymorphic DNA fingerprints at the genomic level with different L. donovani isolates. The results indicate that the PCR and arbitrarily primed PCR (AP-PCR) may be extremely useful approaches for identifying and distinguishing Leishmania parasites.     

Phosphoramidate oligonucleotides as potent antisense molecules in cells and in vivo

Antisense oligonucleotides are designed to specifically hybridize to a target messenger RNA (mRNA) and interfere with the synthesis of the encoded protein. Uniformly modified oligonucleotides containing N3'-P5' phosphoramidate linkages exhibit (NP) extremely high-affinity binding to single-stranded RNA, do not induce RNase H activity, and are resistant to cellular nucleases. In the present work, we demonstrate that phosphoramidate oligonucleotides are effective at inhibiting gene expression at the mRNA level, by binding to their complementary target present in the 5'-untranslated region. Their mechanism of action was demonstrated by comparative analysis of three expression systems that differ only by the composition of the oligonucleotide target sequence (HIV-1 polypurine tract or PPT sequence) present just upstream from the AUG codon of the firefly luciferase reporter gene: the experiments have been done on isolated cells using oligonucleotide delivery mediated by cationic molecules or streptolysin O (SLO), and in vivo by oligonucleotide electrotransfer to skeletal muscle. In our experimental system phosphoramidate oligonucleotides act as potent and specific antisense agents by steric blocking of translation initiation; they may prove useful to modulate RNA metabolism while maintaining RNA integrity.     

Trypanosoma rangeli: discrimination from Trypanosoma cruzi based on a variable domain from the large subunit ribosomal RNA gene

306-314. Three synthetic oligonucleotides corresponding to sequences within the D7a divergent domain of the large subunit ribosomal RNA gene have been used to amplify the total DNA of Trypanosoma rangeli and Trypanosoma cruzi, two morphologically similar protozoa with overlapping geographical distribution and hosts. The two organisms may be distinguished by the electrophoretic mobilities of their respective amplification products. For T. rangeli a 210-bp product was obtained. The presence of this fragment was confirmed in 14 T. rangeli strains. For T. cruzi two possible amplification products were originated: a 265-bp DNA fragment for strains typed as lineage 1 and a 250-bp fragment for lineage 2 strains. Eleven unidentified trypanosome stocks, recently isolated from Amazonian vectors, could be discriminated using the proposed assay. The potential field application of multiplex PCR was further demonstrated by identification of the two parasite species in samples containing intestinal tract and feces of triatomines. In the present study we have also amplified the D7a domain of several trypanosomatids employing primers complementary to the conserved flanking regions. Size and sequence polymorphisms were observed, indicating that this region could also be explored as a target for specific detection of other members of the Trypanosomatidae family.