Site-directed inhibition of DNA replication by triple helix formation.

Sequence-specific DNA recognition can be achieved by the use of triplex-forming molecules, namely, oligonucleotides (TFO) and peptide nucleic acids (PNAs). They have been used to regulate transcription or induce genomic DNA modifications at a selected site in cells and, recently, in vivo. We have determined the conditions under which a triplex structure can inhibit DNA replication in cells. An oligopyrimidine.oligopurine sequence suitable for triplex formation was inserted in a plasmid on both sides of the SV40 origin of replication. This insert-containing plasmid was replicated in COS-1 cells together with the parent plasmid, and the ratio between the corresponding replicated DNAs was quantitated. Selective inhibition of replication of the insert-containing plasmid can be ascribed to ligand binding to the oligopyrimidine.oligopurine sequence. Inhibition of DNA replication was observed using triplex-forming molecules that induce either covalent binding at the double-stranded target sequence (with TFO-psoralen conjugate and irradiation) or noncovalent triplex formation after strand displacement (with bis-PNA). In contrast, in the absence of covalent cross-linking, TFOs (which have been shown to arrest transcription elongation) did not act on replication. These results open new perspectives for future design and use of specific inhibitors of intracellular DNA information processing.

Targeting neighbouring poly(purine.pyrimidine) sequences located in the human bcr promoter by triplex-forming oligonucleotides.

Most poly(purine.pyrimidine) [poly(R.Y)] sequences in eukaryotic genomes are interrupted by one or more base pair inversions. When the inversions are centrally located, the poly(R.Y) sequences can be regarded as the sum of two abutting sites, each potentially capable of forming a triple helix. Employing band-shift, footprinting and modeling methods we examined the formation of triple helices at a critical 27 bp poly(R.Y) sequence interrupted by two adjacent CG inversions, and located in the promoter of the human bcr gene at transcription initiation. We designed several 13-mer and 14-mer triplex-forming oligonucleotides (TFOs) capable of binding the bcr abutting sites, thereby generating different base juxtapositions at the triple helical junction, to examine whether triplex formation occurs in a cooperative manner. It is found that in 50 mM Tris/HCl, pH 7.4, 10 mM MgCl2, 2 mM spermine, 37 degrees C, the 13-and the 14-mer TFOs bind to one half of the bcr site with Delta G between -30 and -35 kJ x mol-1. However, when different 13-mer/14-mer combinations of TFOs were directed against the abutting poly(R x Y) sites, triplex formation has been found to be enhanced only for the triple helical junction formed by the 5'-A-T-3' base juxtaposition, in keeping with a partial stacking suggested from modeling analysis. On the other hand, a longer 24-mer TFO, binding noncooperatively to the same abutting sites, forms a much more stable triplex (Delta G = -51 kJ x mol-1), notwithstanding the two T x CG triads in the middle. Modeling investigations reveal that there is no continuity or propagation of base stacking involving adjacent bases of the third strand at the site of base inversion as well as on the 5' side. The data indicate that the entropy penalty of forming a triplex with two oligonucleotides is much higher than the energy gained from base stacking interactions at the triplex junction formed between the two TFOs.

Anti-gene effect in live cells of AG motif triplex-forming oligonucleotides containing an increasing number of phosphorothioate linkages.

The murine Ki-ras promoter contains a unique polypurine--polypyrimidine [poly(R.Y)] sequence between -290 and -320 from the 3' boundary of exon phi. Previously we demonstrated triplex formation and transcription inhibition promoted by GT and AG oligonucleotides directed against this site [Alunni-Fabbroni et al. (1996) Biochemistry 35, 16361--16369]. In this work, we have investigated triplex formation and anti-gene activity of five 20-mer AG motif triplex-forming oligonucleotides specific for the Ki-ras poly(R.Y) target, derived from 5'-AGGGAGGGAGGAAGGGAGGG (20AG) by replacing an increasing number of phosphodiester linkages with phosphorothioate linkages (S(i)-20AG; i = 2, 3, 4, 5, 19). Electrophoretic mobility-shift experiments (EMSA) showed that four thioate oligonucleotides, S(i)-20AG (i = 2, 3, 4, 5), recognized the Ki-ras target and exhibited dissociation constants similar to that of 20AG: K(d) = 12 +/- 2 nM, while the all-thioate S(19)-20AG exhibited a K(d) of 128 +/- 15 nM. Moreover, the binding between the Ki-ras promoter and oligonucleotides S(i)-20AG (i = 2, 3, 4, 5, 19) was characterized by DMS/piperidine and DNase I footprinting experiments. We observed that the introduction in the phosphodiester oligonucleotide 20AG of sulfur atoms reduced its aggregation significantly and increased its nuclease resistance. Transient transfection experiments using preformed triplexes with a recombinant plasmid containing the reporter chloramphenicol acetyltransferase (CAT) gene under the control of Ki-ras promoter showed that oligonucleotides S(i)-20AG (i = 2, 3, 4, 5, 19) promote a strong inhibition of up to 75% of the CAT expression when compared with control Ki-ras unspecific oligonucleotides. Taken together, these data provide a guideline for designing triplex-forming effector molecules capable of controlling Ki-ras expression in vivo.

Alpha-anomeric configuration of GT oligodeoxynucleotide leads to loss of the specific aptameric and cytotoxic properties retained by the beta-anomeric analog.

The development of antisense, antigene, or aptameric oligonucleotides to modulate in vivo cellular functions depends on using stable biologic molecules. Previous investigations showed that GT oligonucleotides could exert a specific, dose-dependent cytotoxic effect on human cancer cell lines. This is tightly related to the ability of these oligomers to specifically bind nuclear proteins, giving a complex of apparent molecular weight of 45 kDa. We demonstrated that with respect to the cytotoxic GT-beta-oligomer, alpha-anomeric GT analog did not alter the growth of the T lymphoblastic CCRF-CEM cell line, although the cells took it up efficiently. In agreement with this, GT-alpha-oligomer did not form the cytotoxicity-related 45-kDa complex with nuclear proteins. These findings likely could be related to the ability of GT-alpha to structure under nondenaturing conditions because of the high number of T in the sequence.

High concentrations of inhibin A and inhibin B in ovarian serous cystadenoma: relationship with oestradiol and nitric oxide metabolites.

Inhibin production has been demonstrated in malignant epithelial ovarian tumours, but secretion of inhibins by benign cystadenoma has not yet been reported. The present study evaluated the concentrations of inhibin A and inhibin B and the relationship with oestradiol and nitric oxide metabolites in fluid collected from benign ovarian serous cystadenomas (n = 15). In addition, follicular fluid samples (n = 14) from women with regular ovulatory cycles undergoing ovarian stimulation for IVF were studied as a reference group. High concentrations of inhibin A (median = 89.3 ng/ml) and inhibin B (median = 116.1 ng/ml) were found in the cystic fluid of ovarian serous cystadenomas. These inhibin concentrations were even higher than in follicular fluid of stimulated follicles (inhibins A and B = 41.2 and 46.8 ng/ml respectively; P: < 0.001), whereas oestradiol was approximately 18-fold lower in cystic fluid than in follicular fluid (median = 34 versus 622 pg/ml, P: < 0.001). In ovarian cysts, the concentrations of inhibin A and oestradiol were inversely correlated (r = -0.678, P: = 0.008). Cystic fluid samples containing the highest concentrations of NO(2)(-)/NO(3)(-) (45-60 micromol/l) had lower inhibin A and higher oestradiol concentrations than those samples containing lower concentrations (10-25 micromol/l) of NO(2)(-)/NO(3)(-). It is concluded that high amounts of dimeric inhibins are present in ovarian serous cystadenoma. The source of inhibins and the determinants of the inverse association of inhibin A with oestradiol and nitric oxide remain to be determined.

Downregulation of c-Ki-ras promoter activity by triplex-forming oligonucleotides endogenously generated in human 293 cells.

Exogenous triplex-forming oligodeoxynucleotides (TFO) have the capacity to modulate in vivo the expression of individual genes. As the administration of TFO to cells is not without problems, we analyzed the possibility of generating them directly in the cell, using specific expression vectors. We constructed three vectors, mU6-GA, mU6-CA, and mU6-CT, that direct the synthesis in human 293 cells of 76-mer CU, GU, and AG motif TFO (rTFO) potentially capable of binding to a critical poly (R x Y) sequence contained in the promoter of the Ki-ras proto-oncogene. The ability of the CU, GU, and AG motif rTFO to interact with the double helix of the c-Ki-ras target was investigated in vitro by footprinting and band-shift experiments, using both synthetic and endogenously synthesized oligoribonucleotides. The human 293 cells were transfected with DNA mixtures containing a plasmid, which bears the reporter chloramphenicol acetyltransferase (CAT) gene downstream from the c-Ki-ras promoter (pKRS-413), as well as an rTFO-generating vector (mU6-GA, mU6-CA, or mU6-CT). As control, the cells were transfected with DNA mixtures containing vector mU6-C1 or mU6-C2. These generated transcripts unable to form triple helices with the poly (R x Y) sequence of the c-Ki-ras promoter. Intracellular synthesis of the 76-mer CU, GU, and AG rTFO by mU6-GA, mU6-CA, and mU6-CT was checked by Northern blot hybridization. Through beta-gal and CAT ELISA immunoassays, we found that the 293 cells transfected with either mU6-GA, mU6-CA, or mU6-CT showed a significant inhibition of CAT expression compared with cells transfected with control plasmids mU6-C1 or mU6-C2. The results of five separate transient transfection experiments showed that endogenous GU and AG rTFO, generated by mU6-CA and mU6-CT, produce, respectively, 40% (+/- 4% SE) and 47% (+/- 8% SE) CAT inhibition, whereas CU rTFO, generated by mU6-GA, produces 38% (+/- 7% SE) CAT inhibition. In conclusion, this study suggests that it is possible to downregulate the expression of an individual gene through the use of recombinant vectors encoding the information for the intracellular synthesis of short triplex-forming RNA strands.

Effect of phosphorothioate modifications on the ability of GTn oligodeoxynucleotides to specifically recognize single-stranded DNA-binding proteins and to affect human cancer cellular growth.

We have previously identified phosphodiester oligonucleotides exclusively made of G and T bases, named GTn, that significantly inhibit human cancer cell growth and recognize specific nuclear single-stranded DNA binding proteins. We wished to examine the ability of the modified GTn oligonucleotides with different degrees of phosphorothioate modifications to bind specifically to the same nuclear proteins recognized by the GTn phosphodiester analogues and their cytotoxic effect on the human T-lymphoblastic CCRF-CEM cell line. We showed that the full phosphorothioate GTn oligonucleotide was neither able to specifically recognize those nuclear proteins, nor cytotoxic. In contrast, the 3'-phosphorothioate-protected GTn oligonucleotides can maintain the specific protein-binding activity. The end-modified phosphorothioate oligonucleotides were also able to elicit the dose-dependent cell growth inhibition effect, but a loss in the cytotoxic ability was observed increasing the extent of sulphur modification of the sequences. Our results indicate that phosphorothioate oligonucleotides directed at specific single-stranded DNA-binding proteins should contain a number of phosphorothioate end-linkages which should be related to the length of the sequence, in order to maintain the same biological activities exerted by their phosphodiester analogues.

Formation of triple helices at irregular poly (R.Y) sites located in critical positions in the human bcr promoter.

Two polypurine sequences interrupted respectively by one and two adjacent pyrimidines have been identified in the promoter of the human bcr gene. Although these targets are irregular they are recognised and tightly bound by AG and GT motif triplex-forming oligonucleotides. Thermodynamic and kinetic data are presented.

Effect of oligomer length and base substitutions on the cytotoxic activity and specific nuclear protein recognition of GTn oligonucleotides in the human leukemic CCRF-CEM cell line.

We have identified phosphodiester oligonucleotides composed of G and T bases, named GTn, which are able to inhibit the cellular growth of human cancer cell lines by recognising specific nuclear proteins. We demonstrated that GTn oligonucleotides require a length of at least 20 nucleotides in order to exert a significant cytotoxic effect and to retain the specific protein binding ability. In addition, we found that GTn cytotoxicity was lost when A or C bases were introduced at either 3' and 5' end or within the GTn sequences.

Reduction of mdr1 gene amplification in human multidrug-resistant LoVo DX cell line is promoted by triple helix-forming oligonucleotides.

We have demonstrated previously that the GT triplex-forming oligodeoxyribonucleotide (TFO) d(TGTGTTTTTGTTTTGTTGGTTTTGTTT), named TFO ID, targeted to a polypyrimidine-polypurine coding sequence located within human multidrug-resistance mdrl gene, specifically and significantly reduced mdrl mRNA levels in the drug-resistant T-leukemic CEM-VLB100 cell line. In this article, we demonstrate that TFO 1D is effective at inhibiting not only transcription but also replication of mdrl genes, leading to a loss of amplified gene copies in the drug-resistant colon adenocarcinoma LoVo DX cell line. In contrast, TFO ID does not alter replication of the constitutive mdrl gene copy in the corresponding parental sensitive LoVo 109 cell line. A specific reduction in mdrl gene amplification levels was also obtained with the pyrimidine TFO d(CTTTTTCTTTTCTTCCTTTTCTTT), named TFO 24TC, directed against the same polypyrimidine-polypurine sequence of the mdrl gene. We suggest that triple helix-forming oligonucleotides might affect the replication of unstable chromosomal elements as amplicons in actively replicating cells by causing a local impairment of DNA polymerase activity. This study lends support to the notion that TFO may be used to reduce gene amplification aiming to control neoplastic progression in cancer cells bearing amplified oncogenes.

Effect of cations on purine.purine.pyrimidine triple helix formation in mixed-valence salt solutions.

The effect of various monovalent, divalent and oligovalent cations on the reaction of triplex formation by GT and AG motif triplex-forming oligonucleotides, designed to bind to biologically relevant polypurine-polypyrimidine sequences occurring in the promoters of the murine Ki-ras and human bcr genes, has been investigated by means of electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments. We found that in the presence of 10 mm MgCl2 the triple helices were progressively destabilized by adding increasing amounts of NaCl, from 20 to 140 mm, to the solution. We also observed that, while the total monovalent-ion concentration was constant at 100 mm, the exchange of sodium with potassium, but not lithium, results in a further destabilization of the triple helices, due to self-association equilibria involving the G-rich triplex-forming oligonucleotides. Potassium was found to destabilize triplex DNA even when the triple helices are preformed in the absence of K+. However, footprinting experiments also showed that the inhibitory effect of K+ on triplex DNA is partially compensated for by millimolar amounts of divalent transition metal ions such as Mn2+ and Ni2+, which upon coordinating to N7 of guanine are expected to enhance hydrogen-bond formation between the target and the third strand, and to reduce the assembly in quadruple structures of G-rich triplex-forming oligonucleotides. Triplex enhancement in the presence of potassium was also observed, but to a lesser extent, when spermine was added to the reaction mixture. Here, the ion effect on triplex DNA is rationalized in terms of competition among the different valence cations to bind to triplex DNA, and differential cation stabilization of unusual quadruplex structures formed by the triplex-forming oligonucleotides.

Impairment of the mucosal immune system: IgA and IgM cleavage detected in vaginal washings of a subgroup of patients with bacterial vaginosis.

The integrity of the immunoglobulins in vaginal washings of patients with bacterial vaginosis was examined to answer the question of the lack of immune response against Gardnerella vaginalis cytolysin. Clinically diagnosed patients (n=100) were recruited and their vaginal washings examined by Western blotting. Many showed IgA and IgM partially or extensively degraded. According to the degradation pattern, the patients were subdivided into 4 subsets, from intact (score 0) to completely degraded IgA (score +3). Statistical analysis of the data showed a correlation between IgA degradation and absence of immune response to G. vaginalis cytolysin. The extent of IgA degradation correlated also with the sialidase (but not with the prolidase) activity level. All women showed intact IgG and human serum albumin and no trypsin-like activity. Patients with bacterial vaginosis having high sialidase activity and extensive IgA degradation in their secretions could incur more dangerous infections and adverse pregnancy outcomes.

Immunoglobulin A response against Gardnerella vaginalis hemolysin and sialidase activity in bacterial vaginosis.

OBJECTIVE: The aim of this study was to investigate the correlation between the immunoglobulin A immune response to Gardnerella vaginalis hemolysin and sialidase activity in vaginal fluids from patients with bacterial vaginosis. STUDY DESIGN: Nonpregnant women who were examined at a gynecologic clinic, in an age range of 18 to 62 years, were enrolled. The study population comprised 131 healthy volunteers, 32 women with bacterial vaginosis that was positive for immunoglobulin A to Gardnerella vaginalis hemolysin, 40 women with bacterial vaginosis that was negative for immunoglobulin A to Gardnerella vaginalis hemolysin, and 19 women with Candida vaginitis. Bacterial vaginosis was diagnosed by clinical criteria and Gram stain. RESULTS: Sialidase activity was present in 75% (54/72) of patients with bacterial vaginosis. Women having bacterial vaginosis and lacking a specific immunoglobulin A response had a significantly higher level of sialidase activity than patients who had an immune response against Gardnerella vaginalis hemolysin. Sialidase activity was detected in 87% (35/40) of the former subgroup of patients with bacterial vaginosis and in 59% (19/32) of women of the latter subgroup. No sialidase activity was measured in patients with candidiasis. Specificity of the assay for healthy controls was 95% (124/131 women without sialidase activity). CONCLUSIONS: Sialidases produced by Prevotella bivia and other microorganisms present in the microflora of patients with bacterial vaginosis are very likely a virulence factor not only by destroying the mucins and enhancing adherence of bacteria but also by impairing a specific immunoglobulin A immune response against other virulence factors such as cytotoxin from Gardnerella vaginalis.

Human cancer cell lines growth inhibition by GTn oligodeoxyribonucleotides recognizing single-stranded DNA-binding proteins.

Oligonucleotides can specifically target not only nucleic acids but also proteins. Some proteins recognizing oligonucleotides in a sequence-specific manner have been related to cancer transformation and progression. We have found that oligonucleotides composed by repeated and/or variable intervals of GTn with 1 < or = n < or = 7, are able to exert a specific and dose-dependent growth inhibition on human CCRF-CEM, CEM-VLB300, U937, Jurkat, H9 and HeLa tumor cell lines. In contrast, G-->C, G-->A, T-->C and T-->A base substituted control oligonucleotides do not significantly alter cellular growth. In all cell lines, a nuclear protein (molecular mass = 45+/-7 kDa), which specifically recognizes GTn, was identified. Our hypothesis is that the formation of the GTn-protein complex in human cancer cell lines may be involved in the growth inhibition effect. In fact, we found that the reduction or lack of cytotoxic effects by GTn in phorbol 12-myristate 13-acetate-treated CCRF-CEM cells and in normal human lymphocytes is paralleled by the simultaneous reduction or lack of GTn-protein complex. Oligonucleotides specifically 'quenching' intracellular protein activities by forming oligonucleotide-protein complexes may be of potential interest in the treatment of human tumors.

Formation of stable DNA triple helices within the human bcr promoter at a critical oligopurine target interrupted in the middle by two adjacent pyrimidines.

Antigene strategies based on the use of triplex-forming oligonucleotides (TFO) as artificial repressors are constrained by the need for genomic targets with a polypurine-polypyrimidine [poly (R.Y)] DNA motif. In this study, we demonstrate that both A/G and G/T motif oligonucleotides recognize and bind strongly to a critical polypurine sequence interrupted in the middle by two adjacent cytosines and located in the promoter of the human bcr gene at the transcription initiation. The interaction between the designed TFO and this irregular poly (R.Y) target has been studied using a number of techniques, including electrophoretic mobility shift assay (EMSA), circular dichroism (CD), DNase I, and dimethyl sulfate (DMS) footprinting. Although CD shows that the 24-mer TFO self-aggregate in solution, they bind to the bcr target at 37 degrees C, forming stable triplexes that do not dissociate during electrophoretic runs performed up to 50 degrees C in 50 mM Tris-acetate, pH 7.4, 10 mM MgCl2, 50 mM NaCl (buffer A). We used EMSA to determine the equilibrium dissociation constants (Kd) for the reaction T <==> D + TFO at 37 degrees C, either in buffer A or in 50 mM Tris-acetate, pH 7.4, 10 mM MgCl2, 5 mM NaCl (buffer B). The triplexes were found to be more stable in buffer B, a behavior that can be rationalized in terms of monovalent and divalent cation competition for binding to DNA. Footprinting experiments showed that the TFO interact with the irregular poly (R.Y) target in a highly sequence-specific way and that the A/G motif oligonucleotide, juxtaposing T to the double CG inversions of the target, formed the most stable triplex (e.g., 1 microM TFO promoted strong footprints at 37 degrees C). These triplexes, except the one containing two A.C.G mismatched triads, are not destabilized under near physiologic conditions, that is, in 50 mM Tris-acetate, pH 7.4, 80 mM KCl, 20 mM NaCl, 2 mM spermidine. Moreover, we found that guanine N7 in T.C.G and guanine N7 in A.C.G are both accessible to DMS and that the first is less reactive than the second. In conclusion, the results of this study indicate that a critical sequence in the human ber promoter may be used as a potential binding site for TFO designed to repress artificially the transcription of the fused bcr/abl gene expressed in leukemia cells.

A kinetic study of triple-helix formation at a critical R x Y sequence of the murine c-Ki-ras promoter by (A,G)- and (G,T) oligonucleotides.

The kinetics of triplex formation between the oligonucleotides d(AGGGAGG-GAGGAAGGGAGGG) (20AG), d(TGGGTGGGTGGTTGGGTGGG) (20GT) and a 29-bp polypurine-polypyrimidine sequence located in the c-Ki-ras promoter (D) was studied by electrophoretic experiments in 50 mM Tris/acetate, pH 7.4, 50 mM NaCl, 5 mM MgCl2. Rates of triplex formation were determined at three different temperatures (20 degrees C, 37 degrees C and 45 degrees C), under pseudo-first order conditions obtained by using the triplex-forming oligonucleotide (TFO) 500-fold in excess over the target duplex (5 nM). Measurements at TFO/target ratios of 20 and 100 were also carried out. At 37 degrees C the pseudo first-order constants, k(obs), were 18.9 x 10(-5) s(-1) for 20AG and 13.0 x 10(-5) s(-1) for 20GT, yielding association half-lives of 1 h and 1.5 h, respectively. Second-order association constants were found to be in the order of 10(2) M(-1) s(-1): these are slightly lower if compared with those measured for triplex formation by polypyrimidine (C,T) oligonucleotides (10(3) M(-1) s(-1)) [Maher, L. J., Dervan, P. B. & Wolf, B. J. (1990) Biochemistry 29, 8820-8826; Xodo, L. E. (1995) Eur. J. Biochem. 228, 918-926; Bates, P. J., Dosanjh, H. S., Jenkins, T. C., Laughton, C. A. & Neidle, S. (1995) Nucleic Acids Res. 23, 3627-3632] but dramatically lower when compared with duplex recombination from complementary strands (10(6) M(-1) s(-1)) [Craig, M. E., Crothers, D. M. & Doty, P. (1971) J. Mol. Biol. 62, 383-401; Pörschke, D. & Eigen, M. (1971) J. Mol. Biol. 62, 361-381]. Dissociation rate constants, k(-1), were indirectly obtained from equilibrium constants (Kd) and found to be, at 37 degrees C, 6.7 x 10(-7) s(-1) and 5.4 x 10(-6) s(-1) for 20AG and 20GT, respectively. From the rate constants obtained at 20 degrees C, 37 degrees C and 45 degrees C we estimated activation energies of triplex formation between D plus 20AG and D plus 20GT of respectively 134 +/- 29 and 88 +/- 21 kJ/mol. Moreover, the activation energies for the reaction of triplex dissociation were 385 +/- 50 kJ/mol for 20AG and 330 +/- 42 kJ/mol for 20GT. Decreasing the TFO/target ratio from 500 to 100 or 20, we observed a concomitant decrease of the association rate, in keeping with the finding that triplex formation occurs through a bimolecular process. We found that the effect of salt on triplex formation is rather complex, as, the addition of 2 mM spermidine boosted the binding rate of 20GT, but slightly reduced that of 20AG; the increase of NaCl from 50 mM to 100 mM or 150 mM decreased the rate of triplex formation. Finally, the biological implications of the kinetic behaviour exhibited by the two triplex-forming oligonucleotides specific for the c-Ki-ras promoter are discussed.

Specific immune response against Gardnerella vaginalis hemolysin in patients with bacterial vaginosis.

OBJECTIVE: Our goal was to study the mucosal host response in bacterial vaginosis by evaluating the presence of a specific immune response elicited against the Gardnerella vaginalis hemolysin in vaginal fluids of patients and by verifying its correlation with usual criteria adopted to diagnose bacterial vaginosis. STUDY DESIGN: A total of 123 white women attending the gynecologic care unit for urogenital complaints or for screening of uterine malignancies (Papanicolaou test) aged from 20 to 60 years, nonmenstruating, were enrolled. Bacterial vaginosis was diagnosed by clinical criteria and a Gram stain score > 6. RESULTS: We performed the determination of the antibody response in vaginal fluid against the hemolysin produced by G. vaginalis, a common agent present in bacterial vaginosis. The purified G. vaginalis toxin was a suitable antigen for detecting the presence of an immune response in the vaginal fluids of patients with bacterial vaginosis regardless of the strain of G. vaginalis present. A specific immunoglobulin A response was detected in 60% of women with overt bacterial vaginosis (Gram stain score > 6) and in 18.5% of women with intermediate vaginal flora (Gram stain score 4 to 6). The specificity of the test was 91%. CONCLUSIONS: We found a correlation between the specific local immune response to G. vaginalis toxin and bacterial vaginosis. The highly purified form of the toxin is able to discriminate disorders from the opportunistic colonization by G. vaginalis.

Guanine-rich oligonucleotides targeted to a critical R . Y site located in the Ki-ras promoter. The effect of competing self-structures on triplex formation.

The promoter of the murine Ki-ras proto-oncogene contains a (C+G)-rich homopurine . homopyrimidine (R . Y) sequence that is essential for transcription activity. We have designed two G-rich oligonucleotides, d(TGGGTGGGTGGTTGGGTGGG) (20GT) and d(AGGGAGGGAGGAAGGGAGGG) (20AG), that have the potential to bind the critical Ki-ras sequence via triplex-helix formation. Band-shift experiments have shown that 20GT binds the Ki-ras R . Y duplex with a delta G value of -40 +/- 5 kJ/mol, while 20AG appeared to have a lower affinity under the experimental conditions adopted: 50 mM Tris/HCl, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 25 degrees C. In the absence of Mg2+, 20GT did not bind to the Ki-ras R . Y target, while 20AG exhibited the same affinity observed in the magnesium-containing buffer. To gain insight into the solution properties of 20GT and 20AG, we have performed several experiments including polyacrylamide gel electrophoresis (PAGE), hydroxyapatite chromatography, ultraviolet absorption melting and circular dichroism (CD). We found that 20AG rapidly self-associates into presumably a duplex, even at low concentration (< 1 microM), while 20GT forms aggregates slowly, a process favoured by high oligonucleotide concentrations (> 25 microM). The critical Ki-ras sequence was inserted in Bluescript KS+, downstream from the T7 promoter, to investigate to what extent 20AG and 20GT, which are directed against the R . Y target, are able to inhibit T7 RNA polymerase transcription, under near-physiological conditions. Transcription experiments conducted in vitro at pH 7.4 have shown that oligonucleotide 20GT produced a remarkable repression of T7 RNA polymerase activity in the concentration range (10-25 microM), whereas 20AG had little effect on transcription. In conclusion, the results of this work together with other data reported in the literature [Olivas, W. M. & Maher, L. J. III (1995) Biochemistry 34, 278-284; Noonberg, S. B., François, J.-C., Garestier, T. & Hélène, C. (1995) Nucleic Acids Res. 23, 1956-1963], demonstrate that G-rich oligonucleotides, in particular (G,A)-sequences, may raise problems for in vivo application due to self-aggregation.