Bioconjugate Chem. 1995, 6, 278-282
278
Selective Binding of Pyrido[2,3d]pyrimidine 2'-Deoxyribonucleoside to AT Base Pairs in Antiparallel Triple Helices Ross H. Durland,: T. Sudhakar Rao, Krishna Jayaraman, a n d Ganapathi R. Revankar* Triplex Pharmaceutical Corporation, 9391 Grogans Mill Road, The Woodlands, Texas 77380. Received November 15, 1994@
Triple helix-forming oligonucleotides (TFOs) offer the potential to specifically modulate expression of gene in a sequence dependent manner. TFOs containing G and T residues that bind to duplex DNA, forming a series of GGC and TAT base triplets, have been well studied. It has been observed that T is relatively nonspecific in that it binds with similar affinity to AT, GC, and CG base pairs. This may significantly reduce the specificity of a given TFO, leading to undesired effects on the expression of genes unrelated to the intended target. We have now prepared 3-(2-deoxy-~-~-erythro-pentofuranosyl)pyrido[2,3-dlpyrimidine-2,7(8H)-dione (PI and incorporated it into TFOs using the solid-support, phosphoramidite chemistry. It has been demonstrated that a limited substitution of P for T in a G-rich 26-mer TFO can improve binding specificity for AT base pairs in antiparallel motif under certain conditions. The specificity exhibited by P is suggestive of base pair specific interactions that influence the binding strength and consequently enhance the potential therapeutic application of TFOs. However, the effect of substitution of P for T is dependent on the binding conditions, as well as the number and position of substitutions.
INTRODUCTION
Triple helix formation by oligonucleotides has been an area of intense investigation since it was first demonstrated in 1987 ( I ) . A number of investigators have shown that under suitable conditions, triplex-forming oligonucleotides (TF0s)l can bind in the major groove of duplex DNA to form a triple helix (2 and references cited therein). Since TFOs bind to duplex DNA in the major groove, they have the potential to interfere with the binding of various proteins. Formation of triplex a t such a site would block access of the protein to the DNA, thus preventing binding (3-5). Binding of TFOs to specific sites in a variety of eukaryotic genes can also inhibit transcription of these genes, either in vitro or in cell culture (5-91, thus acting as synthetic, site-selective transcriptional repressors. Gene expression is known to be regulated by the actions of a variety of proteins, many of which act by binding to DNA sequences. It has been documented that expression of certain genes is critical for the progression of many diseases, especially viral and malignant diseases. The ability to design a TFO that would bind to a specific sequence and shut off (or turn on) a particular gene could have enormous benefits for the treatment of such diseases. Two factors that are critical for developing TFOs as therapeutics are stability and specificity of triplex fonnation. Stability relates to the strength of the threestranded complex. Obviously, TFOs that bind with high affinity to their target sequences will be more effective than those that bind weakly. Specificity refers to the relative affinity of the TFO for sequences other than the intended target and determines (at least in part) whether a TFO will have unexpected effects on expression of unrelated genes. We have previously shown that G in
* To whom correspondence should be addressed. Tel: (713) 363-8761. Fax: (713) 363-1168. 'Current address: Gene Medicine, Inc., 8301 New Trails Drive, The Woodlands, TX 77381. Abstract published in Advance ACS Abstracts, April 1,1995. Abbreviations: P, pyrido[ 2,3-d lpyrimidine-2'-deoxynucleoside; TFO, triplex-forming oligonucleotide. @
1043-1802/95/2906-0278$09.00/0
the third strand is highly specific for GC base pairs in the target duplex (10). However, binding of T to a target duplex to form a antiparallel triplex is relatively nonspecific. Although binding of T to AT base pairs to form a canonical T.AT triplet is favored, binding to GC and CG base pairs is significant (10). As a result, discrimination between related duplex targets is poor. As part of a program to improve triplex formation through selective chemical modification of TFOs, we have explored various alternatives to T that may provide increased affinity or selectivity in binding to AT base pairs in a target duplex. One base analog that has been examined in this regard is pyridopyrimidine [3-(2-deoxy~-~-erythro-pentofuranosyl)pyrido[2,3-~]pyrimidine-2,7(8H)-dione, 1, PI (11). It has been shown by Ohtsuka et al. (11) that P forms a stable Watson-Crick base pair with G and a less stable wobble base pair with A within double-helical DNA. Our preliminary modeling studies indicated that P can form Hoogsteen hydrogen bonding with AT base pair in the antiparallel triplex motif. The presence of multiple hydrogen-bonding groups on P and its extended ring system may afford increased stacking interactions with neighboring bases in the third strand of the triplex. Thus, we have prepared TFOs containing P and studied their affinity and specificity for AT base pairs. During the preparation of this paper, a paper appeared in which the sequence specificity of P in parallel triplex motif is described (12).The results are strikingly similar to the results reported in this paper. EXPERIMENTAL PROCEDURES
General. The IH NMR spectrum was recorded a t 400 MHz on a Bruker AM400 spectrometer. The 31PNMR spectrum was recorded a t 161.98 MHz on the same spectrometer. Chemical shifts are reported in parts per million downfield from tetramethylsilane (IH, internal) or 85%phosphoric acid (31P, external). Elemental analysis was performed by Quantitative Technologies, Inc., White House, NJ. Reagent grade chemicals were used without further purification unless noted. 0 1995 American Chemical Society
Selective Binding to AT Base Pair
Bioconjugate Chem., Vol. 6,No. 3, 1995 279
Scheme 1. Preparation of Pyrido[2,3-dlpyrimidine Nucleoside DMT phosphoramidite (3)
otides were analyzed by gel electrophoresis to confirm the purity and expected lengths of the oligomers. n For nucleoside composition analysis (131, 0.2-0.3 OD260 units of oligomer was incubated with 2 units of P1 nuclease and 1.5 units of bacterial alkaline phosphatase (Boehringer Mannheim) in 100 pL of 30 mM NaOAc (pH 5.3),1 mM ZnS04 a t 37 "C for 12 h. The pH of the mixture was adjusted to 8.5 by the addition of 20 p L of 0.5 M Tris, and the samples were incubated for a n additional 2 h a t 37 "C. Twenty-five to 75 pL of the digested sample was injected onto a C18 reversed-phase 0 HPLC column, and the components were separated using ti6 CN a gradient of acetonitrile in 50 mM KHzPO4. Peak 2 retention times and U V spectra were compared to those Y 3 of known standards (corresponding to the nucleosides expected for a given oligomer). The calculated relative Synthesis of 3-[5-0-(4,4-Dimethoxytrityl)-2-deoxy- ratios of the nucleosides were in good agreement with ~ - ~ - e r y t h r o - p e n t o f u r a n o s y l l p ~ d o [ 2 , 3 d l p the - experimental ratios indicating that P was successfully 2,7(8N)-dione 3'-O-(Z-cyanoethyl)-N,iV-diisopropyl- incorporated using conventional phosphoramidite chemphosphoramidite (3). The precursor 3-[5-0-(4,4'istry. dimethoxytrityl)-2-deoxy-~-~-erythro-pentofuranosyl]- Analysis of Triplex Formation. In vitro triplex pyrido[2,3-dlpyrimidine-2,7(8H)-dione(2) was prepared formation was assayed using the gel mobility shift as reported by Inoue et al. (11)by tritylation of 1 (Scheme method, essentially as described previously (14,15). 1). Conversion of 2 to the corresponding phosphoramidite Briefly, trace concentrations of radiolabeled (32P)syn(3)proceeded as follows: Compound 2 (0.58 g, 1 mmol) thetic duplex (0.1 x M) were mixed with increasing and N,N-diisopropylethylamine(0.70 mL, 4 mmol) were concentrations of TFO to MI. The standard dissolved in anhydrous CHzCl2 (8 mL). 2-Cyanoethyl binding buffer was 20 mM Tris-HC1, pH 7.6, 10 mM N,N-diisopropylchlorophosphoramidite (0.28 mL, 1.3 MgClZ, and 10% sucrose. Samples were incubated a t 37 mmol) was added under an argon atmosphere and stirred "C for 18-24 h (except as noted) and electrophoresed on for 30 min a t room temperature. The reaction mixture 12% polyacrylamide gels buffered with 89 mM Tris, 89 was diluted with EtOAc (100 mL), and the organic layer mM boric acid, 10 mM MgClz (final pH 8.3). Electrowas washed with saturated NaHC03 solution (30 mL). phoresis was performed a t room temperature a t 3-5 The organic layer was separated, dried (Na2SOd, and Vlcm. The gels were dried and autoradiographed. Apevaporated under reduced pressure. The residue was parent dissociation constants for a given TFOaduplex purified by silica gel column chromatography using CHZinteraction were estimated to be equal to the TFO C1z:EtOAc:NEts (45:45:10, vlv) as the eluent. The fracconcentration required to bind 50% of the labeled duplex tions containing the pure product were pooled, concen(15). trated under reduced pressure, and precipitated into RESULTS pentane a t -30 "C to yield a colorless powder. The powder was collected by filtration and dried under Comparison of P.AT and T-AT Triplets in Antivacuum to yield 0.60 g (77%)of 3. 31PNMR (CD3CN): 6 parallel Triplexes. Table 1presents the initial binding 149.68, 149.81. IH NMR (CD3CN): 6 1.00-1.25 (m, 12 data (acquired a t 20 "C) for pyridopyrimidine (P) in H, isopropyl), 1.93 (m, 2 H, CH of isopropyl), 2.43-2.76 antiparallel triplexes. As shown, P binds to AT base (m, 6 H, 2'Hz and OCHZCHZCN), 3.59 (m, 2 H, ~ ' H z 3.74 ), pairs with substantial affinity, but shows little or no (s, 3 H, OCHd, 3.75 (s, 3 H, OCHd, 4.18 (m, 1 H, 4'H), binding to TA, GC, or CG base pairs. In comparison, T 4.67 (m, 1H, 3'H), 5.90 (d, 1H, vinylic proton), 6.14 (m, binds with substantial affinity to AT, but also binds well 1 H, l'H), 6.45, 6.48 (2d, 1 H, vinylic proton), 6.85 (m, 4 to CG and GC pairs, resulting in a relative lack of H, DMT), 7.23-7.45 (m, 9 H, DMT), 8.68 and 8.70 (25, 1 specificity. It should be noted, however, that binding of H, N J f l . Anal. Calcd for C42H48N508P.0.75 HzO: C, T to AT appears to be slightly better than binding of P 63.42; H, 6.27; N, 8.81. Found: C, 63.19; H, 6.75; N, 8.78. to AT. Figure 1 shows a schematic of a possible P-AT Oligonucleotide Synthesis and Characterization. base triplet. P can exist in two tautomeric forms, Figure All oligonucleotides used in this study were synthesized 2 (12). Both tautomers (P1 and P2) are capable of employing standard solid-support, phosphoramidite chembinding to AT base pairs. It appears that tautomer P1 istry on an Applied Biosystems Model 380B or 394 that carries a proton a t N-8 may bind in antiparallel automated DNA synthesizer. In order to minimize orientation and the tautomer P2 that carries a proton a t potential degradation of the novel nucleoside under N-1 may bind in parallel orientation. It is not known standard deprotection conditions (30% NH40H, 56 "C, whether both tautomers are present or one is predomi16 h), TFOs containing P were prepared using the 2-Nnant over the other. I t may also be possible that the (dimethylformamidine 5'-0-DMT-3'-phosphoramidite of duplex base pair may have a n influence on one tautomer dG (Applied Biosystems) in place of standard dG phosover the other. phoramidite. All other oligonucleotides were prepared When binding studies were repeated a t 37 "C, a using standard P-cyanoethyl phosphoramidites from Mildifferent profile was obtained (Table 2). Under these ligen. Deprotection was carried out in 30% NH40H a t conditions, the oligomer containing T (2102-56) binds to room temperature for 16 h. Under these conditions, no the duplex containing AT base pairs (ZRY102-7) with detectable degradation of the oligomers was observed. about 10-foldhigher affbity than the oligomer containing P (2102-79). Furthermore, the specificity of the interacCrude deblocked oligonucleotides were purified by anion tion between P and AT was reduced a t 37 "C. Discrimiexchange chromatography on a Q Sepharose column equilibrated with 10 mM NaOH, using a gradient of 0.5nation by 2102-79 between the intended target, ZRYlO21.5 M NaC1, and desalted by passage through a C18 Sep7, and the mismatched target ZRY100-0 was about 100Pack (Waters) column. Aliquots of purified oligonuclefold a t 20 "C, but only about 20-fold a t 37 "C. Although
ANAO,
280 Bioconjugate Chem., Vol. 6,No. 3, 1995
Durland et al.
Table 1. Comparison of T and P in Antiparallel Triplexes at 20 "C"
ZRY102-8 2102-56 2102-79 ZRY100-0 2 x 10-8
2102-56 2102-79
>1 x 104
ZRY 102-0
1 x 10-8 ,1 x 104 a Triplex incubations were in 20 mM Tris-HC1, pH 7.6, 10 mM MgC12, 10% sucrose. Samples were incubated at -20 "C for 3-5 h prior to electrophoresis.
Figure 1. Hypothetical hydrogen bonding interaction between pyridopyrimidine (P) and a standard duplex AT base pair. 0
0
I
I
dR
dR
P1
P2
Figure 2. Tautomeric forms of P.
subtle changes in binding conditions are expected to alter the absolute affinity of any given complex, we observe changes in relative affinities of P and T as well. At present, we do not know what factors account for the differences in relative binding. One factor may be that G-rich oligonucleotides are known to have substantial secondary and tertiary structure under certain ionic conditions (16-18). It is possible that the observed interaction between oligomer and duplex is complicated by temperature dependent conformation transitions in the free oligomers. Although most of the studies on G-tetrad formation employ medium containing alkaline cations (Na+ or K+), examples of higher order structure of G-rich oligonucleotides in the presence of Mg2+are also known (19, 20). Further Evaluation of P-AT Triplets. Binding of the oligonucleotides containing P was assessed by the gel mobility shift method as outlined in the Experimental Procedures. The results of these analyses are presented in Table 3. We first examined the binding of oligomer 2102-103, in which all of the T residues in 2102-56 are replaced with P. As shown in Table 3, binding of this oligomer to the ideal target, ZRY102-7, did not result in
Table 2. Comparison of T and P in Antiparallel Triplexes at 37 'Ca duplex
TFO
ZRY102-7 2102-56 2102-79
sequence 5'-ccccttccctccttcctccttctccc-3' 3'-ggggaagggaggaaggapgaagaaga¶gg-S'
apparent Kd(M)
5 x 10-10 5 x 10-9
ZRY102-8 2102-56 2102-79
n1 x 10" .1 x 106
2102-56 2102-79
1 x 10-8 1 x 10-7
2102-56 2102-79
2 x 109 1 x 10"
ZRY100-0
zRYI02-0
a
Conditions were identical to those used in Table 1,
Bioconjugafe Chem., Vol. 6,No. 3, 1995 281
Selective Binding to AT Base Pair Table 3. Binding Studies of Pyridopyrimidine in the ZlOO/Z102 System at 37 "Ca duplex ZRY102-7
TFO
sequence
apparent K d ( M )
5'-CCCCttCCCtCCttCCtccttctccc-3~
)'-g~ggaagggaggaaggaggaagaggg-5' 2102-56 2102-79 2102-103
ZRYl00-0 2100-50 2102-78 2102-80 ZRY101-0 2100-50 2102-78 ZRY101-1 2100-50 2102-78 ZRYlOl-2 2100-50 2102-78
a
Conditions were identical t o those used in Table 2.
any apparent triplex formation a t TFO concentrations up to 3 x M. These data demonstrated that substituting all nine Ts in 2102-56 with Ps (2102-103) reduced the binding affinity significantly. On the basis of our observation that the binding affinity is considerably reduced a t 37 "C, it was not surprising that substitution with nine Ps resulted in significant reduction in binding affinity. Staubli and Dervan (22)have reported that PP stacking had the least favorable energy of base stacking. 2-102-103 contains three PP stackings. P could also promote the secondary structure of G-rich TFO resulting in reduced triplex formation. We prepared several additional TFOs with P substituted a t varying positions. Comparison of the binding of 2100-50, 210278, and 2102-80 indicated that the affinity of P for different base pairs can be strongly influenced by the sequence context (Table 3). In all cases, however, P-AT triplets appeared to be substantially weaker than TeAT triplets (e.g., compare binding of 2102-78 and 2100-50 to ZRY100-0). DISCUSSION
The data obtained in this study are of considerable interest in the field of triple helical DNA. Our group is one of several that is currently studying triplex-forming oligonucleotides (TFOs) as potential human therapeutics (21). TFOs have been shown to bind in a sequencespecific manner to targets in eukaryotic promoters (24, 22). Under ideal conditions, binding occurs with high affinity and, in the case of antiparallel triplexes, a t physiological pH (14,15). Several reports have claimed that TFOs are capable of modulating gene expression in cultured cells (7, 8). However, much remains to be done to develop this technology to its full potential. One question that has largely been ignored is that of specificity. Although several groups have demonstrated that TFOs can bind to their intended targets with high affinity, few systematic studies on binding specificity have been described. Recently, we found that under certain conditions, antiparallel triplex formation can be
less specific than is desirable (IO). In particular, we found that in some sequences, T in the third strand is unable to effectively discriminate between AT, GC, and CG base pairs in a duplex target. This leads to a situation where a single TFO may bind with comparable affinity to several related target sequences. One major attraction of triplex technology is the potential ability to alter gene expression in a highly sequence dependent manner. Thus, the potential loss of specificity when using T in the third strand is a significant drawback. We hoped that substituting pyridopyrimidine (PI for T in antiparallel TFOs would improve binding specificity with little effect on affinity for the intended target. The present data suggest that such an observation appears to be valid only a t lower temperature with limited P substitution a t selected positions. At high incubation temperatures, it becomes apparent that P-AT triplets are clearly weaker than T-AT triplets. This effect becomes more pronounced when a larger percentage of T residues are replaced with P. Our results are in agreement with that of Staubli and Dervan (12). P may be the only example of a nonnatural nucleoside studied so far that has shown similar results in both parallel and antiparallel triplex formation. The sequence specificity toward AT base pairs and the effect of base stacking on triplex stability are striking in both cases. The ease with which P can participate in parallel as well as antiparallel triplex formation suggests that both tautomers (Figure 2) may be involved in triplex formation with equal effkiency. Aside from the binding properties of P, it is a n attractive base because of its strong fluorescent properties (22). TFOs containing P may be useful for measuring triplex formation by the quenching of fluorescence. It will also be worthwhile to study the effect of P on the secondary structure of G-rich TFOs to understand the loss in binding affinity when P is substituted.
Durland et al.
282 Bioconjugate Chem., Vol. 6,No. 3, 1995 LITERATURE CITED (1) Moser, H. E., and Dervan, P. B. (1987) Sequence-specific cleavage of double helical DNA by triple helix formation. Science 238, 645-650. (2) Thuong, N. T., and HelBne, C. (1993) Sequence-specific recognition and modification of double-helical DNA by oligonucleotides. Angew. Chem,., Znt. Ed. Engl. 32, 666-690. (3) Maher, L. J., 111, Wold, B., and Dervan, P. B. (1989) Inhibition of DNA binding proteins by oligonucleotide-directed triple helix formation. Science 245, 725-730. (4) Blume, S. W., Gee, J . E., Shrestha, K., and Miller, D. M. (1992) Triple helix formation by purine-rich oligonucleotides targeted to the human dihydrofolate reductase promoter. Nucleic Acids Res. 20, 1777-1784. (5) Duval-Valentin, G., Thuong, N. T., and Helene, C. (1992) Specific inhibition of transcription by triple helix forming oligonucleotides. Proc. Natl. Acad. Sei. U.S.A. 89, 504-508. (6) Le Doan, T., Perrouault, L., Praseuth, D., Habhoub, N., Decout, J.-L., Thuong, N. T., Lhomme, J., and Helene, C. (1987) Sequence-specific recognition, photocrosslinking and cleavage of DNA double helix by an oligo-[a]-thymidylate covalently linked to a n azidoproflavine derivative. Nucleic Acids Res. 15, 7749-7760. (7) Postel, E. H., Flint, S. J., Kessler, D. J., and Hogan, M. E. (1991) Evidence that a triplex-forming oligodeoxyribonucleotide binds to the c-myc promoter in HeLa cells, thereby reducing c-myc mRNA levels. Proc. Natl. Acad. Sci. U.S.A. 88, 8227-8231. ( 8 ) Orson, F. M., Thomas, D. W., McShan, W. M., Kessler, D. J., and Hogan, M. E. (1991) Oligonucleotide inhibition of IL2Ra mRNA transcription by promoter region collinear triplex formation in lymphocytes. Nucleic Acids Res. 19, 3435-3441. (9) Maher, L. J., 111, Dervan, P. B., and Wold, B. (1992)Analysis of promoter-specific repression by triple-helical DNA complexes in a Eukaryotic cell-free transcription system. Biochemistry 31, 70-81. (10) Durland, R. H., Rao, T. S., Revankar, G. R., Tinsley, J. H., Mvrick. M. A.. Seth. D. M.. Ravford. J.. Sineh. P.. and Jayaraman, K. (1994fBinding of T a n d T analogsto CG base pairs in antiparallel triplexes. Nucleic Acids Res. 22, 32333240. (11) Inoue, H., Imura, A., and Ohtsuka, E., (1985) Synthesis and hybridization of dodecadeoxyribonucleotides containing
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