©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Gene Expression by Triple Helix Formation in Hepatoma Cells (*)

(Received for publication, May 5, 1995; and in revised form, September 6, 1995)

Guang-Chou Tu (§) Qing-Na Cao Yedy Israel

From the Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The aim of this study was to selectively inhibit human mitochondrial aldehyde dehydrogenase (ALDH(2)) gene expression by triple helix assembly. Eight 21-mer oligodeoxyribonucleotides were designed to bind to two purine-rich sequences in the 5`-flanking region of the human ALDH(2) gene. Gel mobility shift assays showed that triplex formation is sequence-specific for the target duplex and the third strand oligonucleotide. In the presence of Mg, but absence of K, triplex-forming oligonucleotides bind to their target sites with apparent dissociation constants (K) in the 10 to 10M range. Potassium cation virtually suppressed the triplex formation of G-C-rich duplex DNA with natural oligonucleotides, but did not prevent triplex formation with phosphorothioate-modified oligonucleotides. Phosphorothioate-modified oligonucleotides were delivered into human hepatoma Hep G2 cells by cationic liposomes. The reduction in ALDH(2) mRNA levels in the cells was determined by the competitive reverse transcription-polymerase chain reaction. One of the phosphorothioate-modified oligonucleotides designed to form an antiparallel triplex with a target in the 5`-flanking region of human ALDH(2) gene (-105 to -125 from the translation initiation codon ATG) reduced by 80-90% the ALDH(2) mRNA levels without affecting albumin mRNA levels. Data suggest that triple-helix formation may provide a means to selectively inhibit hepatic ALDH(2) gene expression for therapeutic use.


INTRODUCTION

Liver aldehyde dehydrogenases (ALDHs)^1 play an important role in the in vivo detoxification of aldehydes. Based on enzymological(1, 2) , metabolism(3, 4) , and human genetics (5) studies, it is believed that the mitochondrial isozyme (ALDH(2)) (6) is mainly responsible for the oxidation of acetaldehyde generated during alcohol oxidation in vivo.

Approximately 40% of Orientals have a defective ALDH(2) (ALDH(2)(-)), which has greatly diminished catalytic activity, while virtually all Caucasians examined thus far have the active liver ALDH(2) (ALDH(2)(+))(7, 8) . Individuals presenting low ALDH(2) activity refrain from excessive drinking, likely due to an aversive reaction caused by elevated blood acetaldehyde levels(9) . The discomfort caused by this effect may be responsible for the low prevalence of alcoholism in subjects who present the ALDH(2)(-) allele in Japan and China(10, 11, 12) . We have recently investigated the relative role of the ALDH(2) deficiency on alcohol consumption in North American-born Canadian and Americans of Oriental origin and found that marked protection against alcohol abuse by this allele also occurs in North America and is independent of the degree of acculturation(13) . ALDH(2)(-) homozygotes are virtual abstainers(13) .

Disulfiram (Antabuse), which inhibits ALDH has been used for treatment of alcoholics for many years(14) . However, disulfiram has a number of side effects(15) . The marked difference in the prevalence of alcohol abuse and alcoholism of ALDH(2)(+) and ALDH(2)(-) individuals suggests that a drug able to specifically inhibit the expression of the normal ALDH(2) gene for prolonged periods could be of value as a therapeutic agent for alcohol abuse and alcoholism.

Oligonucleotide-directed triplex formation of double-stranded DNA has been shown to selectively inhibit gene transcription and subsequent protein synthesis in both in vitro(16, 17, 18, 19, 20, 21, 22) and in ex vivo systems(23, 24, 25, 26, 27, 28) . However, other investigators have been unable to demonstrate triplex formation at any of several different G-rich, polypurine DNA target under physiological potassium ion concentrations. Studies revealed that high potassium ion concentrations, such as those found within cells, can virtually suppress the antiparallel triplex formation by G-rich oligonucleotides (29, 30) .

Milligan et al. (29) used 7-deaza-2`-deoxyxanthosine to replace thymidine in a triplex-forming oligonucleotide and found that the substitution of 7-deaza-2`-deoxyxanthosine for T causes a greater than 100-fold increase in affinity in the presence of 140 mM KCl for this sequence. Gee et al. (31) and Olivas et al.(32) independently reported that site-selective substitution of 2`-deoxy-6-thioguanosine for guanosine (G) in the triplex-forming oligonucleotide results in an increase in triplex formation in the presence of physiological levels of potassium ion.

We sought another way to enhance the ability of oligonucleotides to form triplexes with their targets under physiological potassium conditions. This report provides evidence that submicromolar concentrations of phosphorothioate-modified oligonucleotides designed to bind to the specific area of 5`-flanking region of human ALDH(2) gene are able to form stable triplex with their corresponding targets under physiological potassium ion concentrations in vitro, and to selectively inhibit the expression of human ALDH(2) gene in human hepatoma Hep G2 cells, without affecting the steady-state albumin mRNA levels.


EXPERIMENTAL PROCEDURES

Synthesis of Oligonucleotides

All natural phosphodiester oligonucleotides (termed as PO-oligos), phosphorothioate-modified oligonucleotides where only three linkages at both 5` and 3` terminus were modified (termed as 5`-PS-3`-oligos) and fully phosphorothioate-modified oligonucleotides (termed as PS-oligos) used in this work were synthesized and purified by the Carbohydrate Research Centre, University of Toronto. Oligonucleotides were sterilized by filtration through a 0.22-µm filter (Millipore). Concentrations were determined spectrophotometrically, using nucleotide molar extinction coefficient at 260 nm. P-End-labeled oligonucleotides were prepared using T4 polynucleotide kinase (Pharmacia Biotech Inc.) and [-P]ATP (Amersham Corp.), followed by NICK spin column (Pharmacia) exclusion chromatography to remove unincorporated label.

Polymerase Chain Reaction (PCR)

PCR was performed as described previously(33) . The following pairs of primers were designed from the sequences of human ALDH(2)(34) and albumin gene(35) . The primers used for amplifications of (i) Target A fragment (400 bp), (ii) Target B fragment (300 bp), (iii) ALDH(2) cDNA fragment (222 bp), and (iv) albumin cDNA fragment (224 bp), respectively, are as follows: (i) 5`-GTCAACTGGGCTCCATTCATTC-3/5`-CCCAATGTGTGCCTTTGACCCC-3`; (ii) 5`-GGCTCAACCAAGGCGAGCTCGT-3`/5`-AGACCTCGGGCTGCTGGTTGGG-3`; (iii) 5`GGCTGGGCTGATAAGTACCAC-3`/5`-CAGGTTGGCCACATAGAGGGC-3`; (iv) 5`-GATGACAACCCAAACCTCCCC-3`/5`-TTTGGCAACAGGCAGGCAGCT-3`.

Gel Mobility Shift Assay

Gel mobility shift analysis was performed according to Durland et al.(16) . Triplex formation was allowed to proceed either under conditions favoring triplex formation (20 mM Tris-HCl buffer, pH 7.4, 10 mM MgCl(2), and 10% sucrose) or under physiological ion concentrations (140 mM K, 10 mM Mg buffered to pH 7.4, and 10% sucrose). Samples containing trace concentrations (10M) of P-end-labeled triplex-forming oligonucleotides were incubated with increasing concentrations of unlabeled 300- or 400-bp fragment bearing the target site at 4 °C overnight. Gel mobility shift analysis was carried out at 4 °C for 1-2 h using 5% nondenaturing polyacrylamide gel electrophoresis. Running buffer contained 89 mM Tris-boric acid, pH 7.4, and 10 mM MgCl(2). At the end of electrophoresis, the gel was dried by a model 583 Gel Dryer (Bio-Rad) and autoradiographied. The concentration of double-stranded fragment at the midpoint of the titration is equivalent to the apparent dissociation constant (K(d)) for triplex formation.

Cell Culture and Oligonucleotide Delivery

Human hepatoma Hep G2 cells (American Type Culture Collection) were cultured in 35-mm tissue culture dishes (Corning) with alpha-minimal essential medium containing 10% heat-inactivated fetal bovine serum and 1:100 diluted PSN antibiotic mixture (Life Technologies, Inc.) at 37 °C, 5% CO(2), 100% humidity.

Oligonucleotides were delivered into cultured Hep G2 cells by cationic liposomes (Lipofectamine, Life Technologies, Inc.) according to the manufacturer's recommendations. Oligonucleotides (0-4 µg) and liposomes (24 µg) were diluted separately in 100 µl of OptiMEM (Life Technologies, Inc.). These two solutions were then gently mixed and incubated at room temperature for 45 min to form oligonucleotide-liposome complex. The cultured cells at an 80% confluence were rinsed twice with OptiMEM prior to the addition of a mixture of 800 µl of OptiMEM and 200 µl of oligonucleotide-liposome complex. The cells were exposed to the complex for 6 h at 37 °C, 5% CO(2), 100% humidity, and then returned to the growth medium. Antibiotics were not present during the liposome-mediated delivery. After 24 h of incubation, the cells were rinsed twice with phosphate-buffered saline (Life Technologies, Inc.) prior to total RNA preparation.

To determine the delivery efficiency of oligonucleotides into Hep G2 cells by Lipofectamine, cells grown in 35-mm culture dishes were washed twice with OptiMEM prewarmed to 37 °C. P-End-labeled oligonucleotides were added to the cells at a final concentration of 1 µM in the absence or presence of 24 µg/ml Lipofectamine. At the indicated times, the medium was removed and the cell monolayer was washed four times with ice-cold phosphate-buffered saline. Cells were detached from the dish and harvested from 0.2 M glycine buffer (pH 2.8). Cell- or medium-associated radioactivity was quantified by liquid scintillation.

Extraction of Total RNA and Proteins

Total cellular RNA was prepared from rinsed cells (see above) using Trizol reagent (Life Technologies, Inc.) according to the manufacturer's instructions. The concentration of RNA was measured by absorbance at 260 nm using a spectrophotometer (Beckman DU-640). Total proteins were prepared by lysing the rinsed cells with 1 N NaOH at room temperature overnight(36) . After 1:100 dilution, the protein concentrations were determined using a MicroBCA protein assay reagent kit (Pierce) according to the manufacturer's instructions.

Quantitation of mRNA

mRNAs were quantitated by the competitive reverse transcription-polymerase chain reaction (RT-PCR) (37) . RT was conducted by using First Strand cDNA synthesis kit (Pharmacia) as described by the manufacturer's protocol. A fixed amount of RT product (cDNA) was mixed with a series of amounts (0.01-100 ng) of human genomic DNA (termed gDNA, from Promega) and then subjected to PCR.

To quantitate the amount of PCR product, [alpha-P]dCTP at a final concentration of 50 µCi/ml was added to PCR master mix without adjusting the concentration of individual dNTPs in the dNTP stock(37) . After amplification, an aliquot of each sample was subjected to electrophoresis on a 2.0% agarose gel, and two bands were seen by ethidium bromide stain: 222 bp from cDNA and 722 bp from gDNA amplification in the case of ALDH(2) mRNA determination (see Fig. 1), or 224 bp for cDNA and 436 bp for gDNA amplification in the case of albumin mRNA determination (data not shown). The labeled bands were cut out and counted. The ratio of gDNA/cDNA PCR products was plotted as a function of the amount of known gDNA. The counts of gDNA fragment was divided by the ratio of gDNA fragment bp/cDNA fragment bp to correct for the greater number of [alpha-P]dCTP incorporation in the large fragment. The point of equivalence (i.e. where there is 1:1 ratio) is where cDNA equals gDNA and represents the concentration of cDNA in the unknown (37) .


Figure 1: Quantitation of mRNA by competitive RT-PCR. A fixed amount of cDNA, which is equivalent to the amount of mRNA generated by reverse transcription, was co-amplified with various amounts of human genomic DNA (termed gDNA, 0.01-100 ng) by PCR. Lanes 1-13, sample tubes; lane 14, 100-bp ladder (Pharmacia).




RESULTS

Selection of Target Sites and Design of Triplex-forming Oligonucleotides

Based on the gene sequence of human ALDH(2) published by Hsu and Yoshida(34) , two purine-rich/pyrimidine-rich tracks (the purine-rich sequences of both tracks are in the antisense strand) were chosen from the 5`-flanking region of the gene as the target sites for triplex formation. Fig. 2shows the target sites chosen for the triplex formation. We chose these fragments for two reasons. First, they are located in the regulatory region of the human ALDH(2) gene. Second, these fragments are purine-rich/pyrimidine-rich tracks, which favor the triplex formation.


Figure 2: Selection and synthesis of target sites for triplex formation. Two target sites (A and B) were chosen from the 5`-flanking region of human ALDH(2) gene(34) . Two fragments bearing each target site were synthesized by PCR using two pairs of primers listed under ``Experimental Procedures.'' The CAAT box, TATA box, initiation codon ATG, and codon for Val 17 are marked in the boxes.



For each target site (A or B), eight different types of triplex-forming oligonucleotides were designed (Table 1). All the oligonucleotides are 21-mer. Oligos A-1 and B-1 have the same sequences but opposite orientation (antiparallel) to the purine-rich strand of Target sites A and B, respectively; oligos A-2 and B-2 have the same sequences and same orientation (parallel) as the purine-rich strands; oligos A-3 and B-3 and oligos A-4 and B-4 use thymidines (T) to replace adenosines (A), and guanidines (G) to replace cytosines (C) in the sequences and have different orientation from each other; oligos A-5 and B-5 and oligos A-6 and B-6 use thymidines (T) to replace both adenosines (A) and cytosines (C) in their sequences; oligos A-7 and B-7 and oligos A-8 and B-8 only use thymidines (T) to replace adenosines (A) in their sequences.



Triplex Formation in Vitro

Triplex formation in vitro was assayed by gel mobility shift analysis. Two 300-400 bp of double-stranded DNA fragments of human ALDH(2) gene, which contain Target sites A or B, were synthesized by PCR as described previously (33) using human genomic DNA (Promega) as template and two pairs of oligonucleotides as primers (see Fig. 2and ``Experimental Procedures''). In order to quantitatively assess the strength of the 21-mer oligonucleotide binding to the corresponding target site contained in a 300-400 bp fragment of human ALDH(2) gene, we determined their apparent dissociation constant (K(d)) by titrating each P-labeled oligonucleotide (10M) with increasing concentrations of unlabeled 300-400-bp double-stranded fragments bearing corresponding target sites as described under ``Experimental Procedures.'' An example of this determination (for PS-oligo A-5) is shown in Fig. 3. Table 2lists K(d) values of all designed oligonucleotides. It can be seen from Table 2that in the presence of 10 mM Mg and absence of K, at pH 7.4, K(d) values of these triplex-forming oligonucleotides obtained by the titration are in the 10 to 10M range. This is in agreement with K(d) values which were found for triplex-forming oligonucleotides targeted to gene promoters of human c-Myc(23) , epidermal growth factor receptor, mouse insulin receptor(16) , human dihydrofolate reductase(38) , and rat neu oncogene(31) .


Figure 3: Determination of the apparent dissociation constant (K) using gel mobility shift analysis. Increasing concentrations of the unlabeled 400-bp double-stranded fragment bearing target site A were incubated with a constant amount of radiolabeled PS-oligo A-5 in a buffer containing 140 mM KCl and 10 mM MgCl(2) as described under ``Experimental Procedures.'' The gel was dried and submitted to autoradiography. The apparent dissociation constant K (3-10 times 10M) occurs at the concentration of duplex where oligonucleotide and triplex are equal as determined by densitometry.





In our study the abilities of the oligonucleotides to form triplex with their respective Target sites A or B are in the following order: oligo 5 > oligo 6 > oligo 3 > oligo 4 > oligo 1 and 2 > oligo 7 and 8. These results indicate that the best triplex-forming oligonucleotides should be those which can lead to GbulletG-C, TbulletA-T, or TbulletC-G pairing and are antiparallel to the purine-rich strand of the DNA target duplex (for example, oligos A-5 and B-5). Change in the orientation of oligonucleotides (in the case of oligos A-6 and B-6) was found to reduce the ability of triplex formation. It was also observed that if C, rather than G, is used to form a triplex with C-G (CbulletC-G) only A (in the cases of oligos A-1, B-1 and A-2, B-2), rather than T (in the cases of oligos A-7, B-7 and A-8, B-8), is permissible in the same oligonucleotide to form a triplex with A-T (AbulletA-T).

To further determine the specificity of triplex formation, an oligonucleotide (PS-oligo A-5) designed for Target site A was incubated with the 300-bp fragment bearing Target site B, and an oligonucleotide (PS-oligo B-5) designed for Target site B was incubated with the 400-bp fragment bearing Target site A as described above. It was found that these two oligonucleotides are not able to form triplex with the 300- or 400-bp fragments containing mismatch target sites (data not shown). Overall, the results above indicate that the triplex formation is highly sequence-specific in term of both the target duplex and the third strand oligonucleotide. In the presence of 140 mM KCl, except for PO-oligo A-5, none of the natural oligonucleotides are not able to form triplexes with the corresponding duplex targets (Table 2).

Triplex Formation by Phosphorothioate-modified Oligonucleotides

The strong suppression of potassium ion on the triplex formation of G-C-rich DNA duplex would make antigene strategy not feasible. In order to circumvent this obstacle, several laboratories have focused on the substitution of non-natural oligonucleotide bases to enhance the triplex formation(29, 31, 32) . However, these non-natural base oligonucleotides are not commercially available at this moment. We explored another option. We reasoned that the substitution of a nonbridging oxygen atoms in phosphate linkages with sulfur would not only reduce repulsion forces between the phosphodiester backbone in the third strand and that in the duplex, but also create distereomers, some of which would favor the antiparallel triplex formation, thus increasing the affinity of oligonucleotides to their target duplexes.

As shown in Table 2, the phosphorothioate modification greatly reduces the interference of triplex formation by potassium ion. The fully and partially phosphorothioate-modified oligonucleotides are able to form triplexes with their corresponding target fragments in the presence of 140 mM KCl, with K(d) values in the 10 to 10M range.

Oligonucleotide Delivery

In the in ex vivo experiments reported thus far, the triplex-forming oligonucleotides or their derivatives were transferred into the cultured cells by endocytosis(24, 25, 27) . A number of techniques have been developed for the delivery of DNA into cells. One of the approaches is the use of liposome as a carrier system. Cationic liposomes have been used for DNA transfection both in vitro and in vivo(39, 40) , and also for antisense strategy(41, 42) . It was reported that cationic liposomes enhanced the cell uptake, activity, and stability of antisense oligonucleotides. After being introduced into the cytoplasm, the oligonucleotides delivered appear to concentrate within the nucleus (41) .

To determine whether cationic liposomes are able to increase the delivery of oligonucleotides into the target cells, 1 µMP-labeled PS-oligo A-5 was exposed to cultured Hep G2 cells in the absence and presence of 24 µg/ml Lipofectamine for various times as described under ``Experimental Procedures.'' Oligonucleotide uptake is markedly enhanced by liposomes. At virtually all times, the cell-associated radioactivity in the presence of liposomes was 10-fold higher than that obtained without liposome (data not shown). After 6 h of treatment in the presence of liposomes, the intracellular concentration of delivered oligonucleotides reached a concentration of approximately 80 pmol/10^6 cells.

Inhibition of ALDH(2) Gene Expression in ex Vivo by Triplex-forming Oligonucleotides

Based on the in vitro results, 4 of the 17 triplex-forming oligonucleotides were chosen to determine whether they are able to inhibit the expression of human ALDH(2) gene in Hep G2 cells. One natural phosphodiester oligonucleotide (PO-oligo A-5), two fully phosphorothioate-modified oligonucleotides (PS-oligo A-5 and PS-oligo B-5), and one phosphorothioate-modified oligonucleotide where only three linkages at both 5` and 3` terminus were modified (5`-PS-3` oligo A-5) were separately delivered into Hep G2 cells by cationic liposomes.

It was reported that cationic liposomes may be toxic to some cell lines (41) , and that oligonucleotides containing four contiguous G residues may be antiproliferative(43) . In our study at the concentration used, neither Lipofectamine nor phosphorothioate-modified oligonucleotides containing contiguous G residues (PS-oligo A-5) affected the total amount of proteins in Hep G2 cultures (data not shown).

The steady-state levels of ALDH(2) mRNA in Hep G2 cells were quantitatively determined after oligonucleotide/liposome complex treatment. As shown in Fig. 4a, both 5`-PS-3` oligo A-5 and PS-oligo A-5 inhibited the expression of human ALDH(2) gene in a dose-dependent manner, while PO-oligo A-5 and PS-oligo B-5 did not (see ``Discussion''). The concentrations of PS-oligo A-5 and 5`-PS-3` oligo A-5 for 50% inhibition are the order of 150 and 300 nM, respectively. As shown in Fig. 4b, triplex-forming oligonucleotides did not affect albumin mRNA levels in the same experiments.


Figure 4: Effect of triplex-forming oligonucleotides on steady-state levels of human ALDH(2) mRNA (a) and albumin mRNA (b) in Hep G2 cells. One unmodified oligonucleotide (PO-oligo A-5) and three oligonucleotides fully or partially phosphorothioate-modified (PS-oligo B-5; 5`-PS-3` oligo A-5 and PS-oligo A-5,) were delivered into cultured Hep G2 cells by Lipofectamine as described under ``Experimental Procedures.'' After treatment, total RNA of each dish was extracted and the steady-state mRNA levels were quantitated by competitive RT-PCR (Fig. 1). The data are plotted as the measured mRNA level relative to that derived from the determination of mRNA in untreated cells (0 nM). Error bars refer to standard deviations of the average values from four experiments. Oligonucleotide concentrations are indicated below the bars. Bars from left to right: empty bar, PS-oligo B-5; squares, PO-oligo A-5; stippled, 5`-PS-3` oligo A-5; cross-hatched, PS-oligo A-5.




DISCUSSION

In this study, two target sites were chosen from the 5`-flanking region of human ALDH(2) gene. In vitro experiments showed that both these two target sites are able to form triplex with their corresponding phosphorothioate-modified oligonucleotides. However, after being delivered into Hep G2 cells, only PS-oligo A-5, which forms triplex with Target site A, inhibited human ALDH(2) gene expression. This suggests that Target site A, but not Target B, is located in a positive regulatory area in human ALDH(2) gene. After these studies had been completed, Dipple et al.^2 reported that the -160 to -75 of the 5`-flanking region of the gene contains several positive regulatory sites. Target A duplex in our study corresponds to -125 to -105 in the 5`-flanking region. Thus, it is possible that PS-oligo A-5 blocked one of these positive regulatory sites. On the other hand, a negative regulatory site was found by Dipple et al. in the -600 to -490 of the 5`-flanking region of the gene, in line with our finding that PS-oligo B-5, which binds to Target B in -536 to -516 of the 5`-flanking area, did not inhibit gene expression. The fact that the latter oligonucleotide did not activate gene expression may be due to its relatively short (21-mer) length, compared to the 110-mer long segment where the inhibitory sites are located. Thus, the negative regulatory site may not be masked by the oligonucleotide.

The natural phosphodiester oligonucleotide PO-oligo A-5 was found not to inhibit human ALDH(2) gene expression (Fig. 4a). This may not only be due to its weaker affinity to Target A than that of the modified counterpart PS-oligo A-5, in the presence of potassium ions (Table 2), but also due to its lability toward intracellular nucleases in the cells(44) .

As reported by other laboratories(29, 30, 31, 32) , we also found that potassium ions strongly suppress the antiparallel triplex formation of G-rich DNA duplex. The mechanism of this inhibition is unclear. Potassium ion is known to promote the formation of inter- or intramolecular guanine tetraplex, species with G-rich oligonucleotides (31, 45, 46, 47) . The formation of these stable structures would reduce the amount of oligonucleotide available to form triplex structure. However, attempts to overcome the suppression by substitution of 7-deaza-2`-deoxyguanosine for G failed(29) . Milligan et al. reported that the oligonucleotides containing 7-deaza-2`-deoxyguanosine, which are capable of triplex formation but not self-association by Hoogsteen base pairing, were equally suppressed by 140 mM KCl as their corresponding G-rich oligonucleotide counterparts(29) . On the other hand, the substitution of 7-deaza-2`-deoxyxanthosine for T causes a greater than 100-fold increase in affinity in the presence of 140 mM KCl(29) . The authors suggested that this substitution could decrease the repulsion forces between the phosphodiester backbone in the third strand and that in the duplex.

Phosphorothioate oligonucleotide is one of the most commonly employed modified oligonucleotide in the antisense strategy (see (48) ). The substitution for one of the nonbridging oxygen atoms of the internucleotide phosphate by sulfur produces a compound that is highly resistant to degradation by nucleases (49) and also creates a new stereogenic center(50, 51) . Phosphorothioate-modified oligonucleotide prepared by existing methodologies consists of the mixture of 2^n diastereomers where n is a number of phosphorothioate internucleotide linkage. This fact results in averaging of many characteristics, which are noticeably different in stereoregular oligomers, especially ``all R(p)'' and ``all S(p).'' The differences are observed in hydrophobicity, hybridization parameters, and charge density(52) . Hacia et al.(53) observed that in the absence of potassium ions, the purine-rich phosphorothioate oligonucleotides were able to form triplex with their corresponding duplex, but the pyrimidine-rich counterparts were not.

Analyses of experimental evidence regarding bond order and charge delocalization in thiophosphate anions have led to the conclusion that the negative charge is localized mainly on the sulfur, which can be represented by the partial valence-bond structure O=P-S (see (54) ). Since O=P-S has less electronegativity than O=P-O, we reasoned that the substitution would reduce the repulsion forces between phosphodiester backbone in the third strand and that in the duplex, thus increasing the affinity of triplex-forming oligonucleotides to their duplex targets.

On the other hand, inspection of molecular models clearly indicates that the P-S bond-axis for the R(p) configuration is oriented into the major groove, whereas in the S(p) configuration this bond-axis points away from helix with sulfur thus being positioned on the face of the sugar-phosphate backbone(54) . It is conceivable that some of phosphorothioate-modified oligonucleotide distereomers may favor the antiparallel triplex formation under physiological conditions. From the observation that the fully phosphorothioate-modified oligonucleotide inhibits the expression of human ALDH(2) gene in a stronger manner than the partially modified one does, it could be concluded that the more substitutions an oligonucleotide has the more would the antiparallel triple-helix formation be favored.

It should be noted that we have not measured transcription rates per se. Therefore, the hypothesis that triplex-forming oligonucleotides act by gene-specific inhibition of transcription initiation has not been answered conclusively. A nuclear run-on assay, which is often used to measure transcription rates, could not be used in this study due to its lesser sensitivity.


FOOTNOTES

*
This work was supported by the National Institute on Alcohol Abuse and Alcoholism and Alcoholic Beverage Medical Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust St., Rm. 275, Philadelphia, PA 19107.

(^1)
The abbreviations used are: ALDH, aldehyde dehydrogenase; PCR, polymerase chain reaction; bp, base pair(s); RT, reverse transcriptase; oligo, oligonucleotide; PO-oligo, phosphodiester oligonucleotide.

(^2)
K. M. Dipple, M. J. Stewart and D. W. Crabb, submitted for publication.


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