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A Psoralen-Conjugated Triplex-Forming Oligodeoxyribonucleotide. Containing Alternating Methylphosphonate-Phosphodiester. Linkages: Synthesis and ...
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Bioconjugate Chem. 1999, 10, 572−577

A Psoralen-Conjugated Triplex-Forming Oligodeoxyribonucleotide Containing Alternating Methylphosphonate-Phosphodiester Linkages: Synthesis and Interactions with DNA Paul S. Miller,* Sarah A. Kipp, and Cory McGill† Department of Biochemistry, School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205 . Received November 18, 1998; Revised Manuscript Received March 2, 1999

A psoralen-conjugated oligodeoxyribopyrimidine (1443), PS-pTTTTCTTTTCTTCTT, where PS is trimethylpsoralen and C is 5-methyl-2′-deoxycytidine, that contains alternating methylphosphonatephosphodiester internucleotide linkages was synthesized. The ability of 1443 to form triple-stranded complexes with a purine tract in a synthetic DNA duplex was studied. Irradiation of solutions containing the DNA target and 10 µM 1443 or 0.25 µM of a similar psoralen-conjugated oligodeoxyribonucleotide that contained all phosphodiester linkages, (1193), with long-wavelength UV light resulted in approximately 80% formation of interstrand cross-links at pH 7.0, 37 °C, in the presence of 20 mM magnesium chloride. The extent of triplex formation as monitored by photo-cross-linking decreased over the pH range 5.5-8.0, and the apparent pK of the 5-methylcytosines (C) in 1443 was approximately one-half of a pH unit less than that of the 5-methylcytosines in 1193. Oligomer 1443 formed triplexes in the absence of magnesium, and maximum triplex formation was observed in solutions containing 2.5 mM magnesium, whereas maximal triplex formation by the fully charged 1193 was not observed until the magnesium concentration was 10 mM or higher. Unlike the allphosphodiester backbone of 1193, the alternating methylphosphonate-phosphodiester backbone of 1193 is resistant to hydrolysis by exonucleases in fetal calf serum. The nuclease resistance of 1443 and its ability to form triplexes at very low magnesium concentrations suggests that triplex-forming oligomers with alternating methylphosphonate-phosphodiester backbones may be good candidates for use as antigene reagents in cell culture.

INTRODUCTION

Short oligonucleotides can bind to double-stranded DNA to form triple-stranded complexes. These triplex forming oligomers or TFOs1 interact with homopurine tracts in DNA through the formation of Hoogsteen or reversed Hoogsteen hydrogen bonds. This observation has generated considerable interest in developing TFOs to suppress or manipulate gene expression (for reviews, see refs 1 and 2 and references therein). Most of the current studies on triplex formation have employed oligodeoxyribonucleotides. While certain oligodeoxyribonucleotides can form stable triplexes under physiological conditions, the phosphodiester linkages of these oligonucleotides are susceptible to nuclease hydrolysis, a property that can potentially limit their use in cell culture and in vivo. This problem can be circumvented in principle by using nuclease-resistant oligonucleotide analogues. A number of nuclease-resistant oligonucleotide analogues have been shown to form triplexes with double-stranded DNA. These include oligodeoxyribonucleotide phosphorothio* To whom correspondence should be addressed. Phone: (410) 955-3489. Fax: (410) 955-2926. E-mail:[email protected]. † Summer Research Intern sponsored by the Leadership Alliance. 1 Abbreviations: d-AOMP, oligodeoxyribonucleotide with alternating methylphosphonate-phosphodiester internucleotide linkages; d-C, 5-methyldeoxycytidine; HPLC, high-performance liquid chromatography; Mops, 3-(N-morpholino)propanesulfonic acid; p, methylphosphonate linkage; TFO, triplex-forming oligonucleotide.

ates (3-6), R-anomeric oligonucleotides (7-9), peptide nucleic acids (PNAs) (10, 11), oligonucleotides with riboacetal internucleotide linkages (12), and oligonucleotides with methylphosphonate (13) or (aminoalkyl)phosphonate internucleotide linkages (14, 15). Previous studies showed that a pyrimidine-containing oligodeoxyribonucleotide with methylphosphonate linkages could form a stable triplex, but only at low pH (13). In an effort to increase triplex stability while maintaining nuclease resistance, we have recently begun to explore triplex formation by chimeric oligodeoxyribonucleotides that contain alternating methylphosphonate-phosphodiester internucleotide linkages, d-AOMPs. In this paper, we describe the interaction of a psoralen-conjugated d-AOMP with double-stranded DNA. MATERIALS AND METHODS

Nucleoside-derivatized controlled pore glass supports, protected deoxyribonucleoside-3′-O-(β-cyanoethyl-N,Ndiisopropyl)phosphoramidites, and 2-[4′-(hydroxymethyl)4,5′,8-trimethylpsoralen]hexyl-1-O-[β-cyanoethyl-(N,Ndiisopropyl)]phosphoramidite were purchased from Glen Research, Inc, Sterling, VA. Protected deoxyribonucleoside-3′-O-(N,N-diisopropyl)methylphosphonamidites were obtained from JBL Scientific, San Luis Obispo, CA. All chemicals were of reagent grade or better. Phosphoramidite and phosphonamidite solutions were prepared using HPLC-grade acetonitrile stored over calcium hydride. Polyacrylamide gel electrophoresis was carried out on 20 × 20 × 0.75 cm gels containing 20% acrylamide and 7 M urea. The running buffer was TBE, which contained 89 mM Tris, 89 mM boric acid, and 0.2 mM

10.1021/bc980140m CCC: $18.00 © 1999 American Chemical Society Published on Web 05/13/1999

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Figure 1. General structure of a psorlen conjugated oligodeoxyribonucleotide containing alternating methylphosphonate/ phosphodiester internucleotide linkages, d-AOMP. The sequences of the triplex forming oligomers d-AOMP 1443 and all diester oligomer 1193 and the DNA target 1196/1197 are also shown.

ethylenediaminetetraacetate buffered at pH 8. The gel loading buffer contained 90% formamide, 0.05% xylene cyanol, and 0.05% bromophenol blue. Syntheses of Oligodeoxyribonucleotides. Oligodeoxyribonucleotides 1196, 1197, psoralen derivatized oligodeoxyribonucleotide 1193, and psorlen-derivatized alternating methylphosphonate-phosphodiester oligonucleotide 1443 whose sequences are shown in Figure 1 were synthesized on nucleoside-derivatized controlled pore glass supports using an Applied Biosystems model 392 DNA/RNA synthesizer. The concentrations of the phosphoramidites or methylphosphonamidites were 0.15 M in anhydrous acetonitrile and the coupling time was 90 s. Tetrazole in acetonitrile was used as the activating agent. The coupling step was followed by a capping step, then iodine oxidation followed by a second capping step. Capping reagent B consisted of a solution of 2.54 g of (dimethylamino)pyridine in 50 mL of anhydrous pyridine. The synthesizer was programmed to remove the last dimethoxytrityl group from oligomers 1196 and 1197. Oligomers 1193 and 1443 were derivatized with psoralen using the psoralen phosphoramidite reagent. Oligomers 1193, 1196, and 1197 were deprotected by treatment with solution of 50% concentrated ammonium hydroxide in pyridine at 55 °C for 6 h. Oligomer 1443 was deprotected by sequential treatment with ammonium hydroxide and ethylenediamine (16). Following deprotection, oligomers 1196 and 1197 were purified by strong anion-exchange HPLC. A 20 OD260 sample of each oligomer was injected onto the column, and the column

was eluted with 18 mL of a linear gradient of 0 to 0.8 M ammonium sulfate in 20% acetonitrile buffered with 1 mM ammonium acetate at pH 6.2. The flow rate was 0.6 mL/min and the column was monitored at 290 nm. Psoralen-conjugated oligomers 1193 and 1443 were purified by reversed-phase HPLC on a C-18 column using a 20 mL gradient of 2 to 40% acetonitrile in 50 mM sodium phosphate buffered at pH 5.8. The flow rate was 1.0 mL/ min, and the column was monitored at 290 nm. The oligomers were then desalted on SEP PAK C-18 cartridges. Oligomer 1193 was completely degraded to pT and p-dC in the expected ratios when incubated with 2 µg of snake venom phosphodiesterase in 10 mM Tris, pH 8.1, and 2 mM magnesium chloride at 37 °C for 16 h. Similar treatment of oligomer 1443 produced d-pTpT, d-pCpT, d-pTpC and pT, where C is 5-methyldeoxycytidine and p is methylphosphonate. The products of these enzyme digestions were analyzed by C-18 reversed-phase HPLC using a 20 mL gradient of 2 to 40% acetonitrile in 50 mM sodium phosphate buffered at pH 5.8. The extinction coefficients of the oligomers were determined by comparing the absorbances of a given amount of oligomer before and after digestion by snake venom phosphodiesterase as previously described (17). The 260 values obtained were oligomer 1196, 225 000; oligomer 1197, 253 000; oligomer 1193, 107 000; and oligomer 1443, 96 500. Triplex Formation and Photo-Cross-Linking Experiments The 5′-hydroxyl groups of oligomers 1196 and

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1197 were each phosphorylated by polynucleotide kinase using γ-[32P]ATP (specific activity 50-200 Ci/mmole). The labeled oligomers were purified on MicroSpin Sephadex G-25 columns (Amersham Pharmacia Biotech Inc). Duplexes were formed by mixing 1 equiv of labeled strand with 1.2 equiv of complementary unlabeled strand in a triplex buffer system containing 100 mM sodium chloride, 0-20 mM magnesium chloride, and 50 mM Mops buffered at the specified pH. The solution was heated at 65 °C for 2 min and then allowed to cool to room temperature. Photo-cross-linking experiments were carried out as follows. Equal volumes of duplex solution and a solution of the triplex forming oligomer in triplex buffer were mixed, and the solution was incubated at a given temperature for 3-16 h. Ten microliter aliquots of the solution were then irradiated with 365 nm UV light for 10 min at 2 °C in borosilicate glass test tubes as previously described (18). The solvents were evaporated in a Speed Vac concentrator, and the residue was dissolved in 5-10 µL of gel loading buffer. The samples were electrophoresed on a 20% polyacrylamide gel run under denaturing conditions. Gels were run at 800 or 1000 V at room temperature until the bromophenol blue dye had migrated ∼15 cm on the gel. The wet gel was wrapped in Saran Wrap and imaged by autoradiography or by a FujiX BAS 1000 phosphorimager. Phosphorimages were quantitated using the Fuji software package to determine the amount of cross-linking. Control experiments showed that the same percentage of photoadduct formation was obtained regardless of whether the samples were irradiated at the temperature of incubation or at 2 °C. Thus, for example, when 0.1 µM 1193 was incubated with duplex 1196/1197 for 16 h at 22 °C and then irradiated for 10 min, 91% cross-linking was observed when irradiation was carried out at 2 °C and 91% crosslinking was observed when irradiation was carried out at 22 °C. Similarly, incubation of 2.5 µM 1443 and 1196/ 1197 for 16 h at 22 °C followed by irradiation gave 86% cross-linking when irradiation was carried out at 2 °C and 86% cross-linking when irradiation was carried out at 22 °C. The lower irradiation temperature was used in the experiments described here to minimize the possibility of evaporation and thus changes in concentration during irradiation. RESULTS AND DISCUSSION

Synthesis and Stability of the Psoralen-Conjugated d-AOMP Oligomer 1443 whose general structure is shown in Figure 1, consists of thymidine and 5-methyldeoxycytidine linked by alternating methylphosphonate-phosphodiester internucleotide bonds. The oligomer is conjugated with trimethylpsoralen through a six carbon linker attached to the 5′-terminal phosphate group. The oligomer was prepared on a controlled pore glass using commercially available protected nucleoside β-cyanoethyl-N,N-(diisopropyl)phosphoramidites and protected nucleoside N,N-(diisopropyl)methylphosphonamidites. The amino group of 5-methyldeoxycytidine was protected with an isobutryl protecting group. The psoralen group was introduced as the phosphoramidite during the last coupling reaction. A control oligomer, 1193, that contains contiguous phosphodiester linkages was synthesized in a similar manner. Protected oligomer 1443 was first treated with ammonium hydroxide for 30 min at room temperature. This treatment removes the isobutryl protecting groups from the 5-methyldeoxycytidines, partially removes the β-

Miller et al.

cyanoethyl groups, and partially cleaves the oligomer from the support. These conditions do not result in hydrolysis of the methylphosphonate linkages. The oligomer was then treated with 50% ethylenediamine for 6 h at room temperature to complete removal of the β-cyanoethyl groups and to completely cleave the oligomer from the support. This sequential deprotection scheme avoids transamination of the 5-methyldeoxycytidines by ethylenediamine (16). Previous studies have shown that the phosphodiester linkages of oligothymidine d-AOMPs can be hydrolyzed by an endonuclease activity found in snake venom phosphodiesterase (19). Similar results were observed for oligomer 1443. Thus, 1443 was incubated with snake venom phosphodiesterase for 18 h at 37 °C, and the products of the reaction analyzed by C-18 reversed-phase HPLC. In addition to the pT, which results from hydrolysis of the 3′-terminal phosphodiester linkage, three dimers, pTpT, pCp T, and pTpC were observed. As expected, the methylphosphonate linkages of the oligomer were not hydrolyzed. The stability of 1443 in cell culture medium containing 10% fetal calf serum was also studied. Because it was not possible to introduce a radioactive label into the oligomer, the fate of the oligomer was followed by C-18 reversed-phase HPLC. In contrast to the behavior observed with snake venom phosphodiesterase, the internal phosphodiester linkages of 1443 appear to be resistant to the 3′-exonuclease activity that is commonly found in most mammalian sera (20). Thus, only the 3′-terminal thymidine was hydrolyzed when 1443 was incubated with culture medium containing 10% fetal calf serum whereas similar incubation of the all phosphodiester oligomer 1193 resulted in approximately 40% hydrolysis of the oligomer after 1 h and complete hydrolysis after 4 h. Interaction with Double-Stranded DNA. The interaction of 1443 and 1193 with double-stranded DNA was studied using the target system show in Figure 1. The target duplex, 1196/1197, which consists of 27 base pairs, contains a 15 nucleotide homopurine tract. The sugar-phosphate backbones of 1443 and 1193 have the same polarity as this purine tract, and both oligomers would be expected to interact with this tract via Hoogsteen hydrogen bonds with the formation of T‚A-T and C+‚G-C triads (21). 5-Methyl-2′-deoxycytidine, C, was used instead of 2′-deoxycytidine because it has a higher apparent pK than dC and thus enables triplex formation to occur at higher pH. The 5′-T-A/5′-T-A sequence at the duplex/triplex junction would be expected to serve as a binding site for the psoralen group conjugated to the 5′-ends of either 1443 or 1193 (22-24). The interaction of phosphodiester oligomer 1193 and duplex 1196/1197 could be detected by conventional melting experiments. Thus, a melting transition due to the dissociation of 1193 from the triplex was readily observable at pH 8.0 (Tm 24 °C) and a single melting transition (Tm 65 °C) due to dissociation of the triplex was observed at pH 6.2 (data not shown). The Tm of the duplex 1196/1197 was 65 °C at both pH 8.0 and 6.2. Similar melting experiments were carried out with methylphosphonate oligomer 1443 and duplex 1196/1197. In contrast to the results obtained with 1193, very broad transitions with small hypochromicity changes were observed for 1443. The midpoints of these transitions appeared to be approximately 45 °C at pH 6.2 and 16 °C at pH 8.0, although the breath the transitions precluded an accurate determination of the melting temperatures. Triplex formation between 1443 or 1193 and duplex 1196/1197 was observed indirectly by irradiation of

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Figure 3. Effect of oligomer concentration on triplex formation. Solutions Mops containing 10 nM to 10 µM 1193 (O) or 1443 (b) and 2.5 nM *p1196/1197 in 100 mM sodium chloride, 20 mM magnesium chloride, 50 mM Mops, pH 7.0, were incubated at 37 °C for 3 h and then irradiated at 2 °C for 10 min and the products analyzed by gel electrophoresis. The percentage of interstrand cross-linked product was determined by phosphorimaging.

Figure 2. Triplex-mediated photoadduct formation. Solutions containing 0.7 µM 1193 (lane 1) or 2.5 µM 1443 (lane 2) and 100 nM *p1197/1196, or 0.7 µM 1193 (lane 3) or 2.5 µM 1443 (lane 4) and 100 nM *p1196/1197 in 100 mM sodium chloride, 20 mM magnesium chloride, 50 mM Mops, pH 7.0 were incubated for 16 h at 22 °C and then irradiated with 365 nm UV light at 2 °C for 10 min. The products of the reaction were analyzed by PAGE on a 20% gel. The mobilities of the labeled duplex strand (S), interstrand cross-linked products (XL), the monoadduct (M), xylene cyanol (XC) and bromophenol blue (P) are indicated at the side of the gel. The products were quantitated by phosphorimaging.

solutions containing the oligomer and [32P]-labeled duplex with long wavelength (365 nm) UV light followed by analysis of the photoproducts by denaturing polyacrylamide gel electrophoresis. Under these conditions, photoproducts can arise only as a consequence of triplex formation between the oligomer and the duplex and only covalent adducts can give rise to mobility shifts on the gel. Figure 2 is an autoradiogram of a gel from such an experiment. The experiment was carried out at 22 °C in the presence of 2.5 µM 1443 or 0.7 µM 1193 with a duplex whose upper, purine-rich strand or lower, pyrimidinerich strand carried a 5′-terminal [32P]phosphate. Both oligomers form triplexes as signaled by the presence of bands on the gel whose mobilities are less than those of single strands, *p1196 or *p1197. The bands of lowest mobility represent complexes formed by interstrand cross-links between the psoralen-conjugated oligomer and the duplex. The observation that essentially the same percentage of photoadduct is observed when either the upper or lower strand of the duplex is labeled is consistent with this interpretation. These interstrand crosslinks most likely result from formation of photoadducts between the two thymidines at the 5′-T-A/5′-T-A

duplex triplex junction and the furan and pyrone rings of the tethered psoralen (24). The bands of intermediate mobility result from monoadduct formation between the psoralen and the labeled strand of the duplex and represent less than 2% of the total photoadduct formation. The results of this experiment demonstrate that the alternating methylphosphonate-phosphodiester backbone is compatible with the formation of triple-stranded complexes. Effect of Oligomer Concentration on Triplex Formation. The dependence of triplex formation on oligomer concentration is shown in Figure 3. These experiments were carried out at pH 7.0 and 37 °C over a oligomer concentration range of 10 nM to 10 µM. Both oligomers undergo extensive triplex formation at 37 °C as monitored by photoadduct formation. However, an approximately seven times higher concentration of 1443 is required to achieve cross-linking comparable to that of 1193 under these conditions. Photoadduct formation by 1193 is observed at nanomolar concentrations and the transition from no photoadduct formation to maximal photoadduct formation occurs over a relatively narrow concentration range. Thus, maximal photoadduct formation is observed by 0.25 µM oligomer and less than 1% photoadduct formation is seen below 0.07 µM 1193. Reduced photoadduct formation by 0.7 µM 1193 is observed at 37 °C (50%) as compared to that observed at 22 °C (88%, see Figure 2). This reduction most likely reflects the reduced stability of the triplex at the higher temperature. The transition from no photoadduct formation to maximal photoadduct formation by 1443 is observed over a much broader concentration range, from 0.1 to 10 µM. Again, a lower percentage of photoadduct is observed at 37 °C (42%) than at 22 °C (81%) in the presence of 2.5 µM 1443. The differences in the amounts of photoadduct formed by oligomers 1193 and 1443 most likely reflect differences in the stability of the triplexes formed by these two oligomers. Oligomer 1443 contains seven methylphosphonate linkages and thus consists of a mixture of 128 diastereoisomers. Molecular modeling experiments suggest that the Rp methylphosphonate linkage should not interfere with triplex formation (25). Preliminary results with a triplex forming oligomer that contains a single

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Figure 4. Effect of pH on triplex formation. Solutions containing 0.1 µM 1193 (O) or 2.5 µM 1443 (b) and 20 nM *p1196/1197 in 100 mM sodium chloride, 20 mM magnesium chloride and 50 mM Mops buffered at pH 5.5, 6.1, 6.5, 7.0, 7.5, or 8.0 were incubated at 37 °C for 3 h and then irradiated at 2 °C for 10 min and the products analyzed by gel electrophoresis. The percentage of interstrand cross-linked product was determined by phosphorimaging.

methylphosphonate linkage suggest that the oligomer with an Rp methylphosphonate linkage forms a more stable triplex than does the oligomer with an Sp methylphosphonate linkage. Thus, the relatively higher concentrations of 1443 required for photoadduct formation may simply reflect the presence of diastereoisomers with high percentages of lower binding Sp methylphosphonate linkages. Effect of pH on Triplex Formation. Formation of Hoogsteen bonded C+‚G-C triads requires protonation of 5-methylcytosine. Thus triplex formation and stability by oligomers that contain this base increases as pH decreases. The effects of pH on triplex formation by 1443 and 1193 were compared as shown in Figure 4. This experiment was carried out at 37 °C using oligomer concentrations which gave comparable levels of photoadduct formation at low pH. Both oligomers formed the same amount of photoadduct over the pH range 5.5-6.5. Above pH 6.5, photoadduct formation dropped off more rapidly for the triplex formed by 1443 and essentially no photoadduct formation was observed at pH 7.5 or higher under these conditions. In contrast, 1193 continued to form photoadducts at pH 7.5 with no photoadduct formation at pH 8.0. Protonation of the three 5-methylcytosines of 1443 or 1193 introduces multiple positive charges into the oligomer. The resulting charge repulsion between the protonated 5-methylcytosines is probably compensated to some extent by the negatively charged phosphodiester groups in the oligomer backbone. The results of the experiment in Figure 4 suggest that the apparent pK of the 5-methylcytosines in oligomer 1443 is approximately a half pH unit less than the apparent pK of the 5-methylcytosines in oligomer 1193. This decrease in apparent pK is most likely the consequence of the decreased net negative charge of the alternating methylphosphonate backbone of 1443 versus that of the all phosphodiester backbone of 1193. This decreased net charge would therefore make it more difficult to protonate the 5-methylcytosines of 1443, resulting in reduced triplex stability as the pH of the medium is increased. Consistent with this explanation is the observation that apparent pK of cytosine bases in the all methylphosphonate oligomer d(CT)8 is approximately 1.2 pH units less than that of the corresponding all phosphodiester oligomer when these oligomers form 2:1 d(CT)8:d(AG)8 triplexes (13).

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Figure 5. Effect of magnesium ion concentration on triplex formation. Solutions containing 0.1 µM 1193 (O) or 2.5 µM 1443 (b) and 20 nM *p1197/1196 in 100 mM sodium chloride, 50 mM Mops buffered at pH 7.0, and 0, 0,5, 1.0, 2.5, 5.0 10, or 20 mM magnesium chloride were incubated at 22 °C for 3 h and then irradiated at 2 °C for 10 min and the products analyzed by gel electrophoresis. The percentage of interstrand cross-linked product was determined by phosphorimaging.

Effect of Magnesium Concentration on Triplex Formation. Triplex stability is sensitive to ionic strength and is stabilized by divalent metal ions such as magnesium. The effect of magnesium ion concentration on photoadduct formation by 1443 and 1193 was examined as shown in Figure 5. These experiments were carried out at concentrations of 1443 and 1193 that gave comparable amounts of photoadduct in the presence of 20 mM magnesium ion. As shown in Figure 5, 1443 formed an appreciable amount of photoadduct even in the absence of magnesium. Maximal photoadduct formation was observed once the magnesium concentration reached 2.5 mM. In contrast to this behavior, very little photoadduct formation was observed for 1193 in the absence of magnesium and maximal photoadduct formation was not observed until the magnesium concentration reached 10 mM. The differences in the dependence of triplex formation on magnesium concentration between the two oligomers can most reasonably be explained by the reduced number of phosphodiester linkages in the backbone of 1443. Because the sugar phosphate backbones of the triplex forming oligomer and the purine strand of the target are in close proximity, triplex formation would be expected to entail considerable charge repulsion between these two strands. This charge repulsion can be reduced by the presence of divalent metal ions or polycations such as spermine. Oligomer 1443 contains 7 negatively charged phosphodiester groups as opposed to the 14 negative charges in the backbone of oligomer 1193. Thus, charge repulsion between the backbone of 1443 and the purine strand of 1196/1197 would be expected to be considerably less than that in the triplex formed by 1193 and consequently the dependence of triplex stability on magnesium concentration is reduced. CONCLUSION

The results of the experiments described above show that an oligopyrimidine with an alternating methylphosphonate-phosphodiester backbone can form stable triplexes with a duplex DNA target under physiological conditions of ionic strength and temperature. Triplex forming oligomers with this type of backbone may have distinct advantages over triplex forming oligodeoxyribonucleotides. Because the backbone of the d-AOMP is

Triplex-Forming Methylphosphonate Oligonucleotide

resistant to nuclease hydrolysis, this type of oligomer may be particularly useful in cell culture experiments. The reduced dependence of triplex formation by this type of oligomer on divalent metal ion concentration may also promote triplex formation in the intracellular environment. A potential drawback of pyrimidine containing TFOs is their pH dependence of triplex formation. This problem becomes more serious as the number of 5-methylcytosines in the oligomer increases and is particularly serious for oligomers that contain multiple contiguous 5-methylcytosines. The problem appears to be more serious for the partially charged d-AOMPs than fully charged oligodeoxyribonucleotides. This problem can in theory be circumvented by use of base analogues which do not require protonation or by employing other triplex motifs such as the antiparallel purine motif to form triplexes. Experiments to test these possibilities are currently in progress. ACKNOWLEDGMENT

The research described in this paper was supported by a grant from the National Institutes of Health, GM57140. A portion of this work was also supported by a grant from the National Cancer Institute, CA42762. LITERATURE CITED (1) Svinarchuk, F., and Malvy, C. (1998) Gene-Targeted TripleHelix-Forming Oligonucleotides. In Applied Antisense Oligonucleotide Technology (C. A. Stein, and A. M. Krieg, Eds.) pp 471-486, Wiley-Liss, New York. (2) Faruqi, A. F., and Glazer, P. M. (1998) Triplex-Forming Oligonucleotides for Genetic Manipulation: An Alternative View. In Applied Antisense Oligonucleotide Technology (C. A. Stein, and A. M. Krieg, Eds.) pp 487-507, Wiley-Liss, New York. (3) Kim, S. G., Tsukahara, S., Yokoyama, S., and Takaku, H. (1992) The Influence of Oligodeoxyribonucleotide Phosphorothioate Pyrimidine Strands on Triplex Formation. FEBS Lett. 314, 29-32. (4) Hacia, J. G., Wold, B. J., and Dervan, P. B. (1994) Phosphorothioate Oligonucleotide-Directed Triple Helix Formation. Biochemistry 33, 5367-5369. (5) Lacoste, J., Francois, J.-C., and Helene, C. (1997) Triple Helix Formation with Purine-Rich Phosphorothioate-Containing Oligonucleotides Covalently Linked to an Acridine Derivative. Nucleic Acids Res. 25, 1991-1998. (6) Joseph, J., Kandala, J. C., Veerapane, D., Weber, K. T., and Guntaka, R. V. (1997) Antiparallel Polypurine Phosphorothioate Oligonucleotides form Stable Triplexes with the Rat R-1 Collagen Gene Promoter and Inhibit Transcription in Cultured Rat Fibroblasts. Nucleic Acids Res. 25, 2182-2188. (7) Liquier, J., Letellier, R., Dagneaux, C., Ouali, M., Morvan, F., Raynier, B., Imbach, J. L., and Taillandier, E. (1993) Triple Helix Formation by alpha-Oligodeoxynucleotides: A Vibrational Spectroscopy and Molecular Modeling Study. Biochemistry 32, 10591-10598. (8) Noonberg, S. B., Francois, J. C., Praseuth, D., Guieyssepeugeot, A. L., Lacoste, J., Garestier, T., and Helene, C. (1995) Triplex Formation with R Anomers of Purine-Rich and Pyrimidine-Rich Oligodeoxynucleotides. Nucleic Acids Res. 23, 4042-4049. (9) Bates, P. J., Laughton, C. A., Jenkins, T. C., Capaldi, D. C., Roselt, P. D., Reese, C. D., and Neidle. S. (1996) Efficient Triple Helix Formation by Oligodeoxyribonucleotides Containing R- or β-2-Amino-5-(2-Deoxy-D-Ribofuranosyl)pyridine Residues. Nucleic Acids Res. 24, 4176-4184. (10) Egholm, M., Buchardt, O., Nielsen, P. E., and Berg, R. H. (1992) Peptide Nucleic Acids (PNA)-Oligonucleotide Ana-

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