A Mild and Efficient Solid-Support Synthesis of Novel Oligonucleotide

Bioconjugate Chem. 1998, 9, 283−291

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A Mild and Efficient Solid-Support Synthesis of Novel Oligonucleotide Conjugates Ivan Habus, Jin Xie, Radhakrishnan P. Iyer, Wen-Qiang Zhou, Ling X. Shen, and Sudhir Agrawal* Hybridon, Inc., 620 Memorial Drive, Cambridge, Massachusetts 02139. Received July 18, 1997; Revised Manuscript Received December 16, 1997

Conjugates of oligodeoxyribonucleotide phosphorothioate (ODN-PS) with folic acid, retinoic acid, arachidonic acid, and methoxypoly(ethylene glycol)propionic acid have been synthesized. The procedure involved the initial solid-phase preparation of 5′-amino-functionalized ODN-PS using N-pent-4-enoylderived (PNT) nucleoside phosphoramidites followed by conjugation of the oligonucleotide either to the ligand acids, using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide as a coupling reagent, or to their corresponding succinimidyl derivatives. Subsequent exposure of the support to aqueous ammonium hydroxide (28%, 2 h, 55 °C) resulted in the release of the fully deprotected ODN conjugates, which were purified by reversed-phase HPLC or by preparative polyacrylamide gel electrophoresis. The identity of the oligonucleotide conjugates was confirmed by MALDI-TOF mass spectral analysis.

INTRODUCTION

Oligodeoxynucleotides (ODNs) are widely used as research tools for inhibiting gene expression and as diagnostic agents and are being developed as potential therapeutic agents (1, 2). In recent years, considerable efforts have been devoted to the synthesis of chemically modified ODNs to improve their stability against nucleases, to enhance their binding affinity with the complementary target, and to facilitate their uptake by cells (37). In this context, phosphorothioate (PS) ODNs in which one of the nonbridging oxygens in an internucleotidic phosphodiester linkage is replaced by sulfur have emerged as a promising first-generation antisense therapeutic. Studies on the in vivo pharmacokinetics in rodents reveal that PS-ODNs are widely distributed in tissues and are slowly cleared primarily by urinary excretion. In the development of antisense ODNs as potential anticancer therapeutics, it would be desirable to achieve high intracellular concentration of the ODN at the tumor site. One approach to achieve this objective is to conjugate oligonucleotides to tissue-specific carriers, which would facilitate “carrier-mediated uptake” of the oligonucleotides. In our earlier studies, we had conjugated ODNs to β-cyclodextrin, and its analogues (8, 9), as well as to adamantane (10) to facilitate cellular uptake of ODNs. In the present study, it was envisioned that the dual objectives of tissue-specific targeting and facilitated intracellular delivery could potentially be achieved by conjugating ODNs to ligands such as folic acid (FA) (1), retinoic acid (RA) (2), arachidonic acid (AA) (3), and methoxypoly(ethylene glycol)propionic acid (PEG-A) (4) (Chart 1). Indeed, several papers have described the facilitated intracellular delivery of proteins (11) and other macromolecules (12) as well as liposomes containing entrapped drugs (13) by folate complexation. Presumably, folic acid-receptor-mediated endocytosis is operative in these cases. Recently, a conceptually similar idea was explored in which antisense ODNs were complexed with FA-polylysine conjugate (14, 15). The regulation of RA* Author to whom correspondence should be addressed [telephone (617) 528-7000; fax (617) 528-7692].

induced proliferation and differentiation of various cancerous cells in vitro has been attempted by the use of antisense ODNs (16, 17), peptide nucleic acids (PNA) (18), stable transformant antisense RNA (19), and antisensetransfected clones (20). Similarly, numerous roles have been suggested for AA and its metabolites in cellular function, and antisense ODNs have been employed in studies aimed at delineating these roles (21, 22). In analogy with these studies, we hoped to achieve tissue-specific targeting and facilitated intracellular delivery of ODN conjugates by receptor-mediated endocytosis mechanism, since the receptors for certain growth factors, vitamins, and hormones are overexpressed in rapidly dividing tumor cells (23-26). Conjugation of ODNs with the lipophilic PEG-A was expected to facilitate their cellular uptake, perhaps by passive diffusion, and reduce their protein binding. PEG is also nontoxic and is a pharmacopoeia-approved excipient for topical and oral dosage forms (27). For our studies, we employed an 18-mer ODN sequence targeted against the RIR subunit of protein kinase A (PKA). The down-regulation of the RIR PKA isoform was reported to cause inhibition of tumor growth in a number of experimental in vivo tumor models (28). RESULTS AND DISCUSSION

Synthesis of the Conjugates. A number of methods have been reported for conjugating ODNs to various ligands (29). Two approaches that are commonly employed are as follows: (a) the attachment of the ligand at the 5′-end of the ODN by incorporating the corresponding phosphoramidite during solid-phase ODN synthesis and (b) postsynthetic conjugation of an appropriately functionalized ODN with a ligand. Initially, we attempted to attach FA to the amino-functionalized ODN 5 (Chart 2) by following a procedure for preparation of liposome-FA conjugates (18). The requisite N-hydroxysuccinimide (NHS) ester 6 of FA (1) was prepared and isolated as reported (18). However, in our hands, attempts to synthesize the FA-ODN conjugate 7, by the reaction between 5 and 6, failed to reveal significant product formation when analyzed by reversed-phase

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284 Bioconjugate Chem., Vol. 9, No. 2, 1998

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Chart 1

Chart 2

HPLC and PAGE (data not shown). In a separate model study, we synthesized the amino-functionalized TT phosphodiester (TT PO) 8 and phosphorothioate (TT PS) 9 dinucleotides. Again, the reaction of 8 and 9 with the NHS ester 6 and analysis of the products by reversedphase HPLC did not show the formation of the expected conjugate in isolable yields, but instead revealed a complex mixture of products (data not shown). We then examined an alternate solid-phase approach in which a support-linked functionalized ODN was coupled to the ligands using 1-[3-(dimethylamino)propyl]3-ethylcarbodiimide (DEC) as a coupling reagent. Since folic acid, arachidonic acid, and retinoic acid are known to be heat- and light-sensitive molecules, mild deprotection conditions had to be employed during the preparation of the requisite ODN conjugates. This precluded the use of nucleosides that carry conventional protecting groups for the synthesis of the ODN conjugates as they require harsh deprotection conditions for their removal. In the present study, we employed N-pent-4-enoyl-

derived (PNT)-protected nucleoside monomers (31). However, nucleosides that carry other base-labile protecting groups, e.g., N-tert-butylphenoxyacetyl (t-PAc) (32), can also be employed. As a model study, we prepared the support-linked 5′-amino-terminated dinucleotides TT PO 8a and TT PS 9a (Scheme 1). The synthesis of 8a and 9a was carried out on a 10 µmol scale in an automated DNA synthesizer. During their synthesis, the oxidation of the internucleotidic phosphite linkage was done using a solution of iodine, and the oxidative sulfurization was achieved using a 2% solution of 3H-1,2-benzodithiole-3one 1,1-dioxide (30) in acetonitrile. For the attachment of the 5′-amino linker, the column was removed from the synthesizer and exposed to an acetonitrile solution of the 5′-amino-modifier phosphoramidite C6. Oxidation and capping, followed by the removal of the monomethoxytrityl (MMT) group, gave the support-linked dinucleotides 8a and 9a. The dimers 8a and 9a were then reacted with a solution of FA in anhydrous DMF in the presence of DEC as the coupling agent for 24 h.

Solid-Support Synthesis of Oligonucleotide Conjugates

Bioconjugate Chem., Vol. 9, No. 2, 1998 285

Scheme 1

Chart 3

After successive washing with DMF and acetonitrile, the support was exposed to aqueous ammonium hydroxide (28%, 2 h, 55 °C) to release the fully deprotected dinucleotide conjugates 10-13 (Chart 3). The crude reaction mixtures were analyzed by HPLC (Figure 1) and purified by preparative HPLC. Although the analytical HPLC chromatograms of the purified conjugates revealed the apparent presence of only one product peak, derived from 8 and 9, the MALDI-TOF mass spectra showed two sets of molecular ions presumably corresponding to mono-

and bisconjugates 10 and 11 (calcd for 10, m/z 1149.0, found, m/z 1147.0; calcd for 11, m/z 1856.6, found, m/z 1854.0), as well as 12 and 13 (calcd for 12, m/z 1181.13, found, m/z 1181.8; calcd for 13, m/z 1920.84, found, m/z 1923.5) derived from 8a and 9a, respectively. Also, the presence of the FA moiety in each of the conjugates 1013 was ascertained by UV-VIS spectra, which revealed the presence of characteristic bands for folic acid chromophore at λ290 to λ390 nm (Figure 2). The formation of the monoconjugates 10 and 12 as well as the bisconju-


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Figure 1. Reversed-phase HPLC profile of the reaction mixture between support-bound 8a (A), 9a (B), and between FA and isolated product mixture of 10 and 11 (C) and 12 and 13 (D). The solid-phase reaction was carried out as follows: A mixture of 8a or 9a on solid support (250 mg of CPG), 1 (0.5 mmol), DEC (2.5 mmol), and anhydrous DMF (10 mL) was shaken at room temperature for 24 h under nitrogen atmosphere. The CPG was filtered off and washed successively with DMF and acetonitrile. The products were released from the solid support, deprotected in ammonium hydroxide for 2 h at 55 °C, and after evaporation to dryness purified by reversed-phase HPLC. HPLC was carried out using a Waters 600E system controller, a 996 photodiode array detector, an NEC PowerMate 486/33i, and a Millenium 2010 chromatography manager. For further details see Experimental Procedures.

Figure 2. UV-VIS spectra of the compounds FA, 8, and a mixture of 10 and 11 (A) and and 1, 9, and a mixture of 12 and 13 (B). UV-VIS spectra were recorded on a Perkin-Elmer Lambda 2 spectrometer.

gates 11 and 13 is consistent with the expected reaction of 8 and 9 with one and two carboxylic functions of FA, respectively. Furthermore, according to the HPLC analysis (Figure 1A,B), the integral ratio of the area under the

respective peaks corresponding to the dinucleotides and the derived conjugates were 1:5 for 8:(10 + 11) and 1:7 for 9:(12 + 13), reflecting an overall conversion of 8085% for the conjugation step.



Chart 4

At this juncture, we did not develop procedures for the separation of the mono- and bisconjugates, but applied the methodology for ODN conjugate synthesis. The principal advantage of this solid-phase approach was that all nucleophilic sites remained fully protected while the nucleophilic amino terminus reacted with the ligands in a chemoselective fashion. Consequently, a relatively high yield of the desired conjugates was obtained by this approach compared to the alternate solution-phase approach. Thus, the corresponding fully protected supportbound ODN PS 5 was synthesized on a 10 µmol scale using PNT-protected nucleoside phosphoramidites, and dCPNT nucleoside was anchored to succinylated controlled pore glass (CPG) on an automated DNA synthesizer. The attachment of the 5′-amino-modifier C6 was performed manually on a solid support. After oxidative sulfurization and capping, the MMT group was removed, and the CPG-bound ODN 5 was reacted with FA, RA, and AA in anhydrous DMF and PEG-A in anhydrous pyridine for 24-72 h. After successive washing with DMF or pyridine and acetonitrile, the support-bound conjugates were treated with aqueous ammonium hydroxide (28%, 2 h, 55 °C) and purified by reversed-phase HPLC or, alternatively, by preparative PAGE, to give the corresponding ODN conjugates 7 and 14-17 (Chart 4). Upon analysis by PAGE, it was found that the conjugation reaction of ODN 5 with FA on solid support gave two products, 7 and 17. The products 7 and 17 were

isolated and examined by MALDI-TOF mass spectrometry. The major product 7 (faster moving band on the PAGE, Figure 3) had one molecule of FA attached to the ODN (found, m/z 6319.1, calcd, m/z 6319.50) (Figure 4A). The minor product 17 (slower moving band on PAGE, Figure 3) had two ODNs bridged with one molecule of FA (MALDI-TOF spectrum showed a broad molecular ion between m/z 11900 and 13500 and centered approximately at m/z 12351.1; calcd, m/z 12197.54). To further ascertain their structural identity, we synthesized the ODN PS 18 (36-mer, twice as long as the sequence 5) as a standard. Upon analysis by PAGE the electrophoretic mobilities of both 17 and the 36-mer 18 were found to be similar (Figure 3). Interestingly, the formation of the bisconjugate 17 could be minimized by reducing the reaction time from 72 to 24 h and/or by replacing the C6 amino linker with the C3 amino linker. This way, we could isolate a single product with C6 amino linker or 19 with C3 amino linker (found, m/z 6277.31, calcd, m/z 6277.40). The crude ODN conjugates 14-16 (derived from RA, AA, and PEG-A, respectively) were also purified by preparative HPLC. The conjugates 14 and 15 showed a single sharp band by PAGE analysis (Figure 3), while the PEG oligoconjugate 16 showed a somewhat dispersed band presumably because the PEG used in the present study was a mixture of polymers with an average molecular weight of 5245. MALDI-TOF mass spectrom-


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Figure 3. PAGE of the oligonucleotide conjugates (lanes): 16 (A), 17 (B), 18 (C), xylenecyanol (D) (marker dye), 15 (E), 14 (F), 7 (G), and 5 (H). The oligonucleotide conjugates (0.2 A260 unit) were analyzed by electrophoresis on a 20% polyacrylamide gel containing 8 M urea and visualized by UV shadowing.

etry analysis revealed the expected mass ions for 14 (found, m/z 6177.4, calcd, 6178.52) (Figure 4B) and 15 (found, m/z 6180.6, calcd, 6182.55) (Figure 4C). A sample of 16 gave a positive ion MALDI-TOF spectrum showing a broad peak unresolved pseudomolecular ion signal between approximately m/z 10000 and 13000 and centered at 11413 (calcd, 11179.07). Figure 5 shows the CGE profiles of the oligonucleotide conjugates 7, 14, 15, 16, 17, and 19. The ODN conjugates 7 and 14-16 were studied for hybridization properties with the complementary RNA (30-mer) (Table 1). Hybridization studies indicated that incorporation of FA slightly increased the stability of the duplex formed (∆Tm ) 1.8 °C) relative to the unconjugated control ODN, while incorporation of RA and AA had a slight destabilizing effect on duplex formation, ∆Tm ) -3.3 and -2.5 °C, respectively. The incorporation of PEG-A caused more significant reduction in binding (∆Tm ) -4.9 °C; Table 1), presumably due to destabilization of the duplex by steric interference by the large polymer such as PEG (MW ∼ 5245). While our work was in progress, a synthetic route for conjugation of ODNs, with cholic acid, FA, lipoic acid, and pantothenic acid, through thioether and disulfide bonds was reported (34), involving the initial multistep synthetic preparation of solid-support derivatized with cysteine. Also, recently, Kranz and co-workers reported successful conjugation of FA to N-terminal amino groups of protein using DEC (35). In conclusion, we have successfully demonstrated a simple and efficient method for ODN conjugation on solid support. The combination of solid-support ODN synthesis by employing PNT-protected (or other suitable baselabile-protected) nucleoside phosphoramidites and conjugation of the ODN on the solid support has the following advantages: (a) The conjugation could be

Figure 4. MALDI-TOF mass spectra of folic acid-ODN conjugate 7 (A), retinoic acid-ODN conjugate 14 (B), and arachidonic acid-ODN conjugate 15 (C). The molecular ions were at 6319.1, 6177.4, and 6180.6, respectively.

performed at essentially neutral conditions. (b) Both the 5′- and 3′-ends of an amino-linked ODN could be conjugated, which is dependent on the use of the 3′- or 5′-phosphoramidites. (c) Because the PNT group can be removed using multiple deprotection strategies ranging from neutral to mildly basic conditions (32), the use of this base-protecting group allows flexibility in the synthesis of ODN analogues, as well as in the structurally diverse molecules that could be conjugated to ODN. (d) Conjugation on solid support facilitated the removal of excess reactants and made it easier to isolate the products. The ODN conjugates, thus synthesized, formed stable duplexes with complementary RNA target. EXPERIMENTAL PROCEDURES

Materials and Methods. MALDI-TOF mass spectrometry was performed using a PerSeptive Biosystems Voyager Elite Biospectrometry research station coupled with a delayed extraction laser desorption mass spectrometer, using hydroxypicolinic acid as a matrix. UV-VIS measurements were recorded on a PerkinElmer Lambda 2 spectrometer. Tm measurements were recorded on a GBC 920 UV-VIS spectrometer, supplied with GBC Thermocell. Oligonucleotides were assembled on CPG, carrying the leader PNT nucleoside (31), using an Expedite Nucleic Acid Synthesis System equipped with an NEC Image 466es and CTX monitor. A Waters 650E Advanced Protein Purification System equipped with a Waters 600E system controller, a Dynamax model UV-C absorbance detector, and 746 data module was used for purification of the oligonucleotides.



Figure 5. CGE profiles of oligonucleotide conjugates 7 (A), 17 (B), 19 (C), 14 (D), 15 (E), and 16 (F).

For the analysis of the oligonucleotides, a Waters chromatographic system equipped with a 600E system controller, a 996 photodiode array detector, an NEC Power Mate 486/33i, and a Millenium 2010 chromatography manager was used. Preparative reversed-phase HPLC

was done using a column (300 mm length, 10 mm diameter) packed with bulk preparative C18 matrix (125 Å, 55-105 µm). For analytical HPLC, a Waters RadialPak cartridge was used. Buffers used were (A) 0.1 M ammonium acetate and (B) acetonitrile/A, 20:80. For


Habus et al.

Table 1. Tm Data of Oligonucleotidesa

a T determinations were done using complementary RNA (5′-ACCGCCGCCAGUGAGGAGGCACGCAGCCUU-3′). b Absorbance vs m temperature profiles were obtained using a 1 µM concentration for each strand, in 1 mL of buffer (100 mM Na+, 10 mM phophate, 0.1 mM EDTA, pH 7.4).

preparative HPLC, a linear gradient was used: 100% A, 0% B for 5 min, 0-20% B for 15 min, 20-35% B for 30 min, 35-100% B for 15 min; flow rate of 10 mL/min. For analytical HPLC, a linear gradient was used: 100% A, 0% buffer B for 2 min, 0-20% buffer B for 30 min, 2060% buffer B for 30 min; flow rate of 1.5 mL/min; detection at λ254 nm. CGE analysis was carried out using the Beckman System Gold Personal chromatograph P/ACE system 2200, equipped with P/ACE UV absorbance detector and eCAP ssDNA 100 gel column, 47 cm in length. The oligoconjugates were electrokinetically loaded onto the column and analyzed by applying a voltage of 14.1 kV for 60-100 min and using tris-borate 7 M urea buffer for elution with the detector set at 254 nm. DNA and RNA reagents, RNA phosphoramidite monomers (32), and the LCAA-CPG for the synthesis of oligodeoxy and oligoribonucleotides were obtained from PerSeptive Biosystems GmbH and CPG Inc., respectively. The 5′-amino modifiersC6 and C3 phosphoramiditessand 3H-1,2-benzodithiole-3-one 1,1-dioxide were purchased from Glen Research and R. I. Chemicals Inc., respectively. PNT phosphoramidites were synthesized and analyzed as described before (31). Folic acid, retinoic acid, and arachidonic acid were obtained from Aldrich Chemical Co. The HPLC grade solvents were obtained from Fisher Scientific Co. PEG-A (MW 5000) was purchased from Shearwater Polymers, Inc., and used as received. Protocol for Oligonucleotide Conjugate Synthesis. The synthesis of the oligonucleotide phosphorothioate (ODN PS, 18-mer) sequence targeted against the RI R subunit of protein kinase A (PKA) (28) was carried out on a 10 µmol scale on a CPG support (40 µmol/g) in conjunction with PNT β-cyanoethyl 3′-phosphoramidites on an automated DNA synthesizer. A 2% solution of 3H1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile was used for PS oxidative sulfurization. The last DMT was manually removed on the synthesizer, and the deblocking solution was thoroughly rinsed out of the column with

acetonitrile. The CPG column was removed from the synthesizer and purged with argon. 5′-Amino modifier C6 or C3 phosphoramidite (250 mg) was dissolved in anhydrous acetonitrile (1 mL) and transferred into a luer tip syringe. The syringe was then attached onto the CPG column with another syringe containing 4 mL of standard activator solution. Both solutions of phosphoramidite and activator were swished back and forth through the column for 5 min. The CPG column was then installed back into the DNA synthesizer and washed with acetonitrile, followed by oxidation and capping. The MMT group was then removed using deblocking solution. After thorough washing with acetonitrile, followed by washing with anhydrous pyridine (20 mL) and acetonitrile, the CPG oligonucleotide was dried overnight in vacuo over P2O5. (a) Oligonucleotide Conjugates of Folic Acid, Retinoic Acid, and Arachidonic Acid. A mixture of CPG oligonucleotide (250 mg), folic acid (1), retinoic acid (2), or arachidonic acid (3) (0.5 mmol), 4-(dimethylamino)pyridine (DMAP) (0.05 mmol), triethylamine (40 µL), DEC (2.5 mmol), and anhydrous DMF (10 mL) was shaken in a septum-fitted round-bottom flask (25 mL) at room temperature for 24-72 h under nitrogen atmosphere. The CPG-oligonucleotide conjugate was filtered off and washed successively with DMF and acetonitrile. (b) PEG-A Oligonucleotide Conjugate. A mixture of CPG-bound oligonucleotide (250 mg), methoxy-PEG succinimidyl propionate (4, 0.1 mmol), and anhydrous pyridine (5 mL) was shaken in a septum-fitted roundbottom flask (25 mL) at room temperature for 24 h under nitrogen atmosphere. The CPG-oligonucleotide conjugate was filtered off and washed successively with pyridine and acetonitrile. The CPG-bound oligonucleotide conjugates were treated with aqueous ammonium hydroxide (28%, 2 h, 55 °C) and evaporated to dryness, and the crude conjugates 7, 17, 19, and 14-16 were purified by reversed-phase HPLC and/or PAGE. Oligonucleotide conjugates 7 (257 A260 units), 17 (55 A260 units), and 19 (157 A260 units) were


purified by PAGE, while 16 (172 A260 units, 30% overall yield) was purified by reversed-phase HPLC. Oligonucleotide-conjugates 14 (195 A260 units) and 15 (90 A260 units) were purified by both techniques to obtain pure samples. LITERATURE CITED (1) Akhtar, S., and Agrawal, S. (1997) In Vivo Studies with Antisense Oligonucleotides. Trends Pharmacol. Sci. 18, 1218. (2) Agrawal, S., and Iyer, R. P. (1995) Modified Oligonucleotides as Therapeutic and Diagnostic Agents. Curr. Opin. Biotechnol. 6, 12-19. (3) Agrawal, S., Ed. (1996) Antisense Therapeutics, Humana Press, Totowa, NJ, and references cited therein. (4) Akhtar, S., Ed. (1995) Delivery Strategies for Antisense Oligonucleotide Therapeutics, CRC Press, Boca Raton, FL, and references cited therein. (5) Sanghvi, Y. S., and Cook, P. D., Eds. (1994) Carbohydrate Modifications in Antisense Research, ACS Symposium Series 580, American Chemical Society, Washington, DC, and references cited therein. (6) Crooke, S. T., and Lebleu, B., Eds. (1993) Antisense Research and Applications, CRC Press, Boca Raton, FL, and references cited therein. (7) Uhlman, E., and Peyman, A. (1990) Antisense Oligonucleotides: A New Therapeutic Principle. Chem. Rev. 90, 543-584. (8) Zhao, Q., Temsamani, J., and Agrawal, S. (1995) Use of Cyclodextrin and its Derivatives as Carriers for Oligonucleotide Delivery. Antisense Res. Dev. 5, 185-192. (9) Agrawal, S., Zhao, Q., and Habus, I. (1997) Cyclodextrin Cellular Delivery System for Oligonucleotides. U.S. Pat. 5,605,890 and 5,615,565. (10) Habus, I., Zhao, Q., and Agrawal, S. (1995) Synthesis, Hybridization Properties, Nuclease Stability, and Cellular Uptake of the OligonucleotidesAmino-β-cyclodextrins and Adamantane Conjugates. Bioconjugate Chem. 6, 327-331. (11) Leamon, C. P., and Low, P. S. (1993) Membrane Folatebinding Proteins are Responsible for Folate-protein Conjugate Endocytosis into Cultured Cells. Biochem. J. 291, 855-860. (12) Leamon, C. P., and Low, P. S. (1991) Delivery of Macromolecules into Living Cells: A Method that Exploits Folate Receptor Endocytosis. Proc. Natl. Acad. Sci. U.S.A. 88, 55725576. (13) Lee, R. J., and Low, P. S. (1994) Delivery of Liposomes into Cultured KB Cells via Folate Receptor-mediated Endocytosis. J. Biol. Chem. 269, 3198-3204. (14) Citro, G., Szczylik, C., Ginobbi, P., Zupi, G., and Calabretta, B. (1994) Inhibition of Leukemia Cell Proliferation by Folic AcidsPolylysine-Mediated Introduction of c-myb Antisense Oligodeoxynucleotides into HL-60 Cells. Br. J. Cancer 69, 463-467. (15) Citro, G., Perrotti, D., Cucco, C., D’Agnano, I., Sacchi, A., Zupi, G., and Calabretta, B. (1992) Inhibition of Leukemia Cell Proliferation by Receptor-mediated Uptake of c-myb Antisense Oligodeoxynucleotides. Proc. Natl. Acad. Sci. U.S.A. 89, 7031-7035. (16) Baldassarre, G., Bianco, C., Tortora, G., Ruggiero, A., Moasser, M., Dmitrovsky, E., Bianco, A. R., and Ciardiello, F. (1996) Transfection with a Cripto Antisense Plasmid Suppresses Endogenous Cripto Expression and Inhibits Transformation in a Human Embryonal Carcinoma Cell Line. Int. J. Cancer 66, 538-543. (17) Manna, B., Ashbaugh, P., and Bhattacharyya, S. N. (1995) Retinoic Acid-Regulated Cellular Differentiation and Mucin Gene Expression in Isolated Rabbit Tracheal Epithelial Cells in Culture. Inflammation 19, 489-502. (18) Gambacorti-Passerini, C., Mologni, L., Bertazzoli, C., le Coutre, P., Marchesi, E., Grignani, F., and Nielsen, P. E. (1996) In Vitro Transcription and Translation Inhibition by Anti-Promyelocytic Leukemia (PML)/Retinoic Acid Receptor R and Anti-PML Peptide Nucleic Acid. Blood 88, 1411-1417.

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A Mild and Efficient Solid-Support Synthesis of Novel Oligonucleotide

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