Bioconjugate Chem. 2001, 12, 523−528
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New TFO Conjugates Containing a Carminomycinone-Derived Chromophore Massimo L. Capobianco,* Marcella De Champdore´,† Luca Francini,‡ Stefano Lena, Anna Garbesi, and Federico Arcamone ICOCEA-CNR, Via Gobetti 101, 40129 Bologna, Italy. Received November 8, 2000; Revised Manuscript Received February 16, 2001
Conjugates obtained by linking the anthracycline intercalating chromophore to triple helix forming oligonucleotides (TFOs) have been used in a physicochemical study of the stability of triple helices with DNA sequences of pharmacological relevance. The intercalating moiety is represented by carminomycinone derivatives obtained upon O-demethylation and hydrolysis of the glycosidic linkage of daunomycin followed by the introduction of an alkylating residue at two different positions. Results of experiments with a polypurinic region present in the multidrug resistance (MDR) gene indicate that the stability of the triple helix is significantly enhanced by replacement of C’s with 5-MeC’s in the TFO sequences tested. The stability is not changed when a 3′-TpT is present in place of a 3′-CpG at the presumed intercalation site of the anthraquinone chromophore. The same carminomycinone derivatives were used for the preparation of conjugates able to form triple helices with the polypurine tract (PPT) present in the human integrated genome of HIV-1 infected cells. Three different TFOs (T4MeCT4MeCC, C2; T4MeCT4MeCCMeCCMeCCT, C6; and T4MeCT4G6, G6) were designed and linked to the anthraquinone moiety. These conjugates showed a significantly enhanced ability to bind the PPT region of HIV with respect to the nonconjugated TFOs.
INTRODUCTION
One promising new technique used to inhibit the expression of genes consists of the formation of a local triple helix able to interfere with the transcription machinery. Local triple helices can be formed by an exogenous oligonucleotide that binds to a DNA in its natural duplex form, providing it has the right recognition pattern for Hoogsteen’s type of hydrogen bonds, i.e., a polypurine tract. Since the pioneering studies of He´le`ne and co-workers, this approach has been known as antigene methodology (1). Even if polypurine tracts of suitable length for selective recognition (14-20 bases) are relatively rare within genomes, they are often present in the control region of genes (indicating, perhaps, a possible natural mechanism of regulation). In general, triple helices have a rather low thermodynamic stability under physiological conditions of pH and temperature. However, stability may be enhanced, inter alia, by tagging an intercalating agent at one or both termini of the triplex-forming oligonucleotide (TFO). Using different derivatives of daunorubicin as intercalating moieties at pH values between 5.5 and 6.8, we have already increased from 16 to 20 °C the melting temperature of a triplex formed by a pyrimidinic T/C oligonucleotide with a DNA duplex featuring a polypurine region present in the hMDR1 gene (2). The first part of the present work studied the effect of replacing cytidines with 5-methyl-cytidines on the stabil* To whom correspondence should be addressed. Phone: +39 051 6398287, Fax: +39 051 6398349, E-mail: capobianco@ area.bo.cnr.it. † Present address: Dip. Chim. Org. e Biochim., Universita ` degli Studi di Napoli “Federico II”, Via Cinthia 26, Monte S. Angelo, 80126 Napoli, Italy. ‡ Present address: Dip. Chim. Org. “A. Mangini” Universita ` degli Studi di Bologna, Via S. Donato 15, 40127 Bologna, Italy.
ity at pH 7.0 of the same triplex used with the daunorubicin-derived TFO conjugates (2). Using the same model system, we also addressed the question of the possible dependence of the triplex stabilizing effect of this type of intercalator upon the base pair sequence of their putative intercalation site. The second part investigated the use of the same type of conjugation for increasing the thermal stability at pH 7.0 of triplexes formed with the polypurine tract (PPT) region of HIV-1 proviral DNA. This region of the proviral DNA is accessible to TFOs within cell nuclei (3, 4), but native oligonucleotides do not lead to stable triplexes under physiological conditions. Hence, some extra binding strength is needed to obtain a significant inhibition of transcription. MATERIALS AND METHODS
General Methods. All chemicals were of commercial analytical grade and used as received. The following abbreviations were used throughout the text: AcOH, acetic acid; DMF, N,N-dimethylformamide; DTT, 1,4-dithio-D,L-threitol (or 1,4-dithioerythritol); TEAA, triethylammonium acetate; TEAB triethylammonium bicarbonate; Tris, tris(hydroxymethyl)aminomethane. HPLC Analysis and Purifications. HPLC analysis and purifications were performed on a Waters 600E Millipore system control equipped with a Waters 484 tuneable absorbance detector as previously described (2). UV-Melting Experiments. Ultraviolet measurements and melting profiles were recorded on a Perkin-Elmer 554 spectrophotometer equipped with a MGW Lauda RC5 thermostat and a MGW Lauda R40/2 digital thermometer; heating was controlled by an electronic device generating a linear gradient of temperature of 0.5 °C/ min. Samples were prepared by dissolving equimolar amounts of the duplex strand in 0.1 M Tris-AcOH buffer, 0.05 M NaCl, 0.01 M MgCl2 at pH 7.0 at a concentration
10.1021/bc000139z CCC: $20.00 © 2001 American Chemical Society Published on Web 06/20/2001
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of 1 or 2 µM per strand. The UV cuvettes were heated to 80 °C for 20-30 min and then slowly cooled (at least in 2-3 h) to room temperature to favor annealing; the conjugates were then added, and the cuvettes were again heated to 60 °C to favor the annealing of the third strand without destruction of the duplex. The cuvettes were then gently cooled to 4 °C. Condensation of moisture on the cell walls, in the range 4-20 °C, was prevented by flushing nitrogen. Fluorescence Experiments. Fluorescence quenching experiments were monitored on a SPEX 3000 spectrofluorometer at room temperature with 1.25 mm slits, 455 and 495 nm filters, λexc ) 470 nm; the emission was recorded in the interval 520-700 nm. Interference of water was electronically subtracted from the recorded spectra. Electrophoretic Mobility Shift Assay. Electrophoresis was run using a 20% polyacrylamide gel in a thermostatic slab gel unit at 10 V/cm. The samples were prepared by mixing 1 µL of a 0.1 mM buffered solution of duplex with 0.8-1.5 µL of a 0.1 mM buffered solution of conjugate-TFO. The samples were then diluted with buffer to 5 µL and allowed to anneal at 15 or 36 °C overnight; 5 µL of a 50% sucrose solution was added, and the 10 µL samples were applied on the gel. A 0.1 M TrisAcOH buffer, 0.05 M NaCl, 0.01 M MgCl2 at pH 7.0 was used both for the preparation of samples and for the run. Gel staining was obtained by soaking the gel with 0.01% stains-all dye in 1/1 (v/v) water/formamide. The gels were then directly digitalized on a PC scanner. Synthesis and Purification of the Oligonucleotides. The oligodeoxynucleotides were prepared on a Pharmacia gene assembler II-plus using the manufacturers’ protocols with commercially available amidites. The oligonucleotides were deblocked with 30% aqueous ammonia (16 h at 50 °C) and then purified by ion exchange on a Sephacel column (Pharmacia), eluting with a linear gradient from 0.05 to 1.5 M TEAB. The fractions were analyzed by HPLC, and those with purity greater than 90% were pooled together, evaporated several times with water, and then converted to the sodium salt by ion exchange with Dowex WX-8 resin in the sodium form. Synthesis and Purification of Conjugates (Series A and B). Oligonucleotides containing the 5′-PS linkage were prepared using a modified (home-tested) protocol on a Pharmacia gene assembler II-plus employing dicyanoethyl-(N,N-diisopropyl)-phosphoramidite. After deblocking and night-standing on DTT (5 mg/mL), the crude compound was precipitated with 1-butanol (10 volumes); the oligonucleotide was then dried, converted to the sodium salt (as above), and lyophilized. The sodium salt of a 5′-PS-oligonucleotide (10 ODU about 75% pure by HPLC) was dissolved in 125 µL of DMF, 20 µL of water, 11 µL of 15-crown-5, 1 mg of anthracycline iodo derivatives (2), and 0.2 mg of DTT. The reaction mixture was kept in a sealed vial for 16 h in a thermostat at 45 °C. The crude mixture was purified from the excess of iodoalkylcarminomycinone by diluting the mixture with 150 µL of water and extracting the unreacted iodo derivative with a 85/15 v/v solution of dichloromethane/ methanol. The pure conjugate was then recovered by purification of the aqueous solution on a reverse-phase silica gel column using a gradient of acetonitrile (0-30%) in a 0.1 M TEAA aqueous buffer. Usually 5-6 ODU of pure conjugate was obtained. RESULTS AND DISCUSSION
Part 1: hMDR1 Target. The sequences of the TFOs and the target duplexes used in part 1 of this study are
Capobianco et al. Table 1. Oligonucleotides (ODNs) Synthesized and Used in the MDR Work (Boldface C indicates 5-MedC)a ODN
entry code
5′ TTT CTT CTT CTT 3′ 5′ TTT CTT CTT CTT 3′ 5′ AGG AGC AAA GAA GAA GAA CTT T 3′ 3′ TCC TCG TTT CTT CTT CTT GAA A 5′ 5′ AGG ATT AAA GAA GAA GAA CTT T 3′ 3′ TCC TAA TTT CTT CTT CTT GAA A 5′
MDR-1 MDR-2 DNA-1 DNA-2
a The evidenced regions encompassing the intercalation site are common to both DNA-2 and PPT duplex.
Figure 1. The two types of conjugates employed. The hexamethylene spacer is attached to the 5′-base of the ODN through a phosphorothioate (PS) linkage (2).
shown in Table 1 , and the structures of the conjugates are presented in Figure 1. Our previous work (2) demonstrated that derivatization of MDR-1 as in the compounds of the type shown in Figure 1 enhances the stability of the triplex with the DNA-1 target (Table 1). In particular, we observed that, while the triplex formed at pH 6.8 by unconjugated MDR-1 exhibited a Tm of 11 °C, the 4-O- and the 6-O-carminomycinone derivatives of the same TFO (MDR-1-A and MDR-1-B) melted at 27 and 31 °C, respectively. The increased stability of the triplex is related to a specific structure including both recognition of the PPT region by the complementary Hoogsteen strand and intercalation of the anthracycline chromophore. This was deduced from observation of the biphasic melting transition and gel mobility shift in electrophoretic experiments, together with the fluorescence quenching typical of the intercalation process. Fluorescence quenching was observed only as a consequence of the triple helix formation. First, to explore the possibility of further enhancing the stability of the triple helix by substituting the cytidines with the 5-methylcytidine’s sequence, MDR-2 was synthesized and linked to the appropriate carminomycinone derivatives, according to the procedure already described (2), to give MDR2-A and MDR-2-B. Second, although the precise position of the intercalation of the tagged molecule in the triplehelical complex remains to be established, the length of the linker allows it to occur either at the interface between the duplex and the triplex region or, at most, one base pair away from it. As DNA-1 exhibits a 3′-CpG next to the said interface, we evaluated the effect of the substitution of this structural element with, for example, a 3′-TpT as in DNA-2. The stability of the triplexes formed by the TFOs and by the conjugates with DNA-1 and DNA-2 was studied by UV-melting experiments, and the results are summarized in Table 2. As expected, replacing the C’s with 5-MeC’s (MDR-2 versus MDR-1) enhanced the stability of the triple helix by more than 12 °C at pH 7.0. The melting data also show that, at least for the conjugates of type A
TFO Conjugates Containing a Carminomycinone
Bioconjugate Chem., Vol. 12, No. 4, 2001 525
Figure 2. Fluorescence quenching of MDR-2-A with increasing equivalents of DNA-1 duplex (left panel) or DNA-2 duplex (right panel). Measurements were made at pH 7.0 and 22 °C. Table 2. Results of the Melting Experiments with TFO MDR-1 and MDR-2 and Conjugates of the Latter Targeting DNA-1 and DNA-2 at pH 7.0 in 100 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 2 µM per Strand Tm triplex (°C) MDR-1 MDR-2 MDR-2-A MDR-2-B a
DNA-1a
DNA-2b
