Methylphosphonate-Modified Oligo-2'-O ... - ACS Publications

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Bioconjugate Chem. 2004, 15, 498−507

3′-Methylphosphonate-Modified Oligo-2′-O-methylribonucleotides and Their Tat Peptide Conjugates: Uptake and Stability in Mouse Fibroblasts in Culture Chrissy E. Prater and Paul S. Miller* Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205. Received January 8, 2004; Revised Manuscript Received February 18, 2004

Antisense oligo-2′-O-methylribonucleotides and their methylphosphonate derivatives show high binding affinities for their complementary targets under essentially physiological conditions. Additionally, the methylphosphonate linkage is resistant to nuclease hydrolysis. Here we show that a single methylphosphonate internucleotide linkage at the 3′-end of an oligo-2′-O-methylribonucleotide is sufficient to prevent degradation by the 3′-exonuclease activity found in mammalian serum. Complexes formed between a cationic lipid, Oligofectamine, and 5′-[32P]-labeled methylphosphonate modified oligo2′-O-methylribonucleotides are taken up by mouse L929 fibroblasts in culture. The extent of uptake appears to be dependent upon the sequence of the oligonucleotide. Examination of lysates of oligonucleotide treated cells by polyacrylamide gel electrophoresis showed that no degradation of the oligonucleotide occurred, even after incubation for 24 h. A fluorescein-derivatized oligomer was shown to localize mainly in the cell nucleus as monitored by fluorescence microscopy. Covalent conjugates of fluorescein-derivatized 3′-methylphosphonate modified oligo-2′-O-methylribonucleotides with Tat peptide, a cell permeating peptide, were also prepared. The Tat peptide was coupled to the 5′-end of the oligonucleotide using either disulfide coupling chemistry or conjugation of a keto derivative of the Tat peptide via a 4-(2-aminooxyethoxy-2-(ethylureido)quinoline group at the 5′-end of the oligonucleotide. Although formation of the Tat peptide conjugates was confirmed by mass spectrometry, the propensity of these oligonucleotides to form aggregates and their apparent high affinity for plastic and glass made the conjugates unsuitable for studies of uptake by cells in culture.

INTRODUCTION

Antisense research has evolved to include many modifications that render chemically synthesized oligonucleotides resistant to hydrolysis by nucleases, while enhancing the binding affinity of these oligonucleotides for their targets. We have recently described the syntheses and binding properties of antisense oligo-2′-O-methylribonucleotides that contain nuclease resistant methylphosphonate internucleotide linkages (1-6). These studies demonstrated that methylphosphonate-modified oligonucleotides have high affinities for their complementary RNA targets under essentially physiological conditions. To further characterize these oligonucleotide analogues, we have studied their uptake by, and stability in, mammalian cells in culture. Here we describe the cationic lipid-mediated uptake of methylphosphonate containing oligo-2′-O-methylribonucleotides by mouse L929 fibroblasts and show they are delivered intact to the cell’s nucleus. We also describe efforts to prepare covalent conjugates of fluorescein-derivatized oligomers with Tat peptide, a peptide derived from the “protein transduction domain” of HIV-1 Tat protein, which has been reported to facilitate cellular uptake of macromolecules including oligonucleotides. EXPERIMENTAL PROCEDURES

Materials. Protected 2′-O-methylribonucleoside-3′-O(β-cyanoethyl-N,N-diisopropyl) phosphoramidites, 5′* To whom correspondence should be addressed. Phone: 410955-3489. Fax: 410-955-2926. E-mail: [email protected].

fluorescein phosphoramidite (6-FAM), 5′ nucleosidederivatized controlled pore glass supports, 4,5-dicyanoimidazole, 1H-tetrazole, Cap Mix A, Cap Mix B, and oxidizing solution were purchased from Glen Research Inc., Sterling, VA. The exocylclic amino groups of the 2′O-methylribonucleoside phosphoramidites mr-C and mr-G were protected with acetyl and isobutryl protecting groups, respectively. Protected 2′-O-methylribonucleoside-3′-O-(N,N-diisopropyl) methylphosphonamidites were purchased from ChemGenes Corp., Ashland, MA. The exocyclic amino groups of the mr-C and mr-G methylphosphonamidites were protected with isobutryl groups. The phosphoramidite and phosphonamidite solutions were prepared using synthesis grade acetonitrile (Fisher) that was dried and stored over calcium hydride. Tat peptide, residues 48-60, with the addition of a Cterminal cysteine (GRKKRRQRRRPPQC), was synthesized by the Johns Hopkins University School of Medicine Protein/Peptide Sequencing Core Facility. MALDI analysis was performed in the AB Mass Spectrometry/Proteomics Facility at Johns Hopkins School of Medicine (www.hopkinsmedicine.org/msf/) with support from a National Center for Research Resources shared instrumentation grant 1S10-RR14702. Mouse L929 fibroblasts were obtained from American Type Culture Collection (ATCC), Manassas, VA. General Methods. Strong anion exchange (SAX) HPLC was carried out using Dionex DNAPac PA-100 analytical (4 × 250 mm) or semiprep (9 × 250 mm) columns purchased from Dionex Corp., Sunnyvale, CA. The columns were eluted with a 20 min (analytical) or

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Oligo-2′-O-methylribonucleotide Methylphosphonates

30 min (semiprep) linear gradient of 0.0 M to 0.5 M sodium chloride in a buffer containing 100 mM Trishydrochloric acid, pH 7.8, and 10% acetonitrile at a flow rate of 1.0 mL/min (analytical) or 1.5 mL/min (semiprep). The columns were monitored at 260 nm for analytical runs, and 290 nm for preparative runs. C-18 reversedphase HPLC was carried out using a Microsorb C-18 column (0.46 × 15 cm) purchased from Varian Analytical, Sunnyvale, CA. Unless otherwise noted, the column was eluted with a 20 mL linear gradient of 2-30% acetonitrile in 50 mM sodium phosphate, pH 7.8, at a flow rate of 1.0 mL/min. Denaturing polyacrylamide gel electrophoresis (PAGE) was carried out on 20 × 20 × 0.75 cm gels containing 20% acrylamide and 7 M urea. The running buffer (TBE) contained 90 mM Tris, 90 mM boric acid, and 0.2 mM ethylenediaminetetraacetate buffered at pH 8. The gel loading buffer contained 90% formamide, 0.05% xylene cyanol, and 0.05% bromophenol blue. Syntheses of Anti-TAR and Anti-RRE Oligonucleotides. The sequences of the oligonucleotides are shown in Table 1. The 5′-phosphate derivative of oligonucleotide 1 (2) was a gift from Dr. Tomoko Hamma; it was dephosphorylated by incubating 0.03 A260 unit of the oligonucleotide with 1 unit of arctic shrimp alkaline phosphatase in 10 µL of buffer containing 10 mM Tris, pH 8.0, and 10 mM magnesium chloride for 60 min at 37 °C. The solution was then heated at 65 °C for 15 min to deactivate the enzyme. The other oligonucleotides were synthesized on an ABI Model 392 DNA/RNA synthesizer. The protected nucleoside methylphosphonamidites and nucleoside β-cyanoethylphosphoramidites were dissolved in anhydrous acetonitrile at a concentration of 0.15 M. The nucleoside methylphosphonamidite solutions were stored over 3 Å molecular sieves 1 h prior to use. The coupling time for nucleoside methylphosphonamidites was 120 s using a 0.25 M solution of 4,5-dicyanoimidazole (DCI) as the activating agent. A “low-water” oxidizing agent consisting of 1.27 g of iodine, 37.5 mL of tetrahydrofuran, 12.5 mL of 2,6-lutidine, and 100 µL of water was used. The capping solutions consisted of Cap A (10% acetic anhydride, 10% pyridine in tetrahydrofuran (v/v)) and Cap B (1.25 g 4-(dimethylamino)pyridine in 50 mL of anhydrous pyridine). The coupling time for nucleoside β-cyanoethylphosphoramidites was 360 s using the synthesis reagents listed above for the methylphosphonamidites, or 120 s using a solution of 1H-tetrazole as the activating agent and standard synthesis reagents purchased from Glen Research Inc. Fluorescein phosphoramidite coupling times were 10 min using standard synthesis reagents. Derivatization of oligonucleotide 4 with the 4-(2-aminooxyethoxy)-2-(ethylureido)quinoline (AOQ) group was performed by Dr. Tomoko Hamma using methods reported previously (5). Deoxyribo and 2′-O-methylribo diester oligonucleotides were deprotected in 0.4 mL of concentrated ammonium hydroxide for 5 h at 65 °C in a 4.5 mL autosampler vial. After cooling, the supernatant was removed, and the support was washed twice with 0.2 mL aliquots of 50% aqueous acetonitrile. The combined supernatant and washings were evaporated to dryness. The methylphosphonate containing oligo-2′-O-methylribonucleotides were removed from the support by incubation in 0.4 mL of concentrated ammonium hydroxide for 2 h at room temperature in a 4.5 mL autosampler vial. The support was washed as above, and the combined supernatant and washings were evaporated to dryness under vacuum at room temperature. The residue was then deprotected with a solution containing 10 µL of water, 22.5 µL of acetonitrile, 22.5 µL of 95% ethanol, and 50 µL of

Bioconjugate Chem., Vol. 15, No. 3, 2004 499

ethylenediamine for 6 h at room temperature. The solution was cooled on ice and neutralized by addition of 600 µL of ice-cold 2 N hydrochloric acid. After neutralization, the solution was diluted into 10 mL of 2% acetonitrile in 50 mM sodium phosphate buffer at pH 5.8 (C-18 buffer A). This solution was loaded onto a C-18 SEP PAK cartridge (Waters Inc.) that had been preequilibrated with 10 mL of acetonitrile, 10 mL of 50% aqueous acetonitrile, and 10 mL of C-18 buffer A. The SEP PAK was washed with 10 mL of water, and the oligomer was eluted with 3 mL of 50% aqueous acetonitrile. The AOQ-derivatized oligonucleotide was deprotected in a solution containing 5 µL of water, 11.25 µL of 95% ethanol, 11.25 of µL acetonitrile, and 25 µL of ethylenediamine for 1.5 h at room temperature and was immediately purified by C-18 HPLC as previously described (5). The remaining oligonucleotides were purified by SAX HPLC. 10-40 A260 units of the crude oligomer was evaporated to dryness then dissolved in 500 µL of SAX buffer A (0.1 M Tris, 10% aqueous acetonitrile). These samples were then injected onto the SAX column, and the column was eluted with a linear gradient of 0-0.5 M sodium chloride for oligomers 1-2mp and 2, 0-0.75 M sodium chloride for oligomer 3, 0-0.6 M sodium chloride for oligomer 2-1mp, 0-0.65 M sodium chloride for oligomer 3-1mp, and 0-1.0 M sodium chloride for fluoresceinated oligomer 3-1mp-F. Oligonucleotide 3-2mp was purified by PAGE on a 20% gel run under denaturing conditions. The oligomer was located by UV shadowing and was isolated by incubating the gel slice in 1 mL of a solution containing 0.1 M ammonium acetate and 20% aqueous acetonitrile overnight at 37 °C. The supernatant was evaporated to dryness and the residue was desalted on a C-18 SEP PAK cartridge as described above. Oligonucleotides (with the exception of oligomer 1) were analyzed by MALDI-TOF mass spectrometry: dT10 (m/z: calcd 2967.5, found 2968.5); 1-2mp (m/z: calcd 4931.9, found 4931.4); 2 (m/z: calcd 5279.0, found 5279.1); 2-1mp (m/z: calcd 5277.0, found 5277.1); 3 (m/ z: calcd 4935.9, found 4936.4); 3-1mp (m/z: calcd 4933.9, found 4934.8); 3-1mp-F (m/z: calcd 5471.0, found 5474.1); 3-2mp (m/z: calcd 4931.9, found 4931.8); 3-2-mp-F (m/ z: calcd 5748.1, found 5750.8); 4 (m/z: calcd 1598.0, found 1598.5); AOQ-4-F (m/z: calcd 2531.5, found 2534.6). Oligonucleotide Stability in Serum. Oligonucleotides were phosphorylated by incubation of 2 × 10-7 mmol oligomer with 5 units of T4 polynucleotide kinase in 10 µL of buffer that contained 60 µM γ-32P-ATP, (specific activity, 100 Ci/mmol), 50 mM Tris, pH 7.6, 10 mM magnesium chloride, and 10 mM mercaptoethanol for 60 min at 37 °C. A 14 µL aliquot of the reaction mixture was diluted with 140 µL of 0.1 M imidazole buffer, pH 6.0, and 16 µL of 1 M 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (Sigma). The solution was incubated at room temperature for 4 h after which 6.4 µL of ethylenediamine and 13 µL of 12 N hydrochloric acid were added. The pH of the solution was adjusted to 7.4 by further addition of hydrochloric acid, and the solution was incubated overnight at room temperature. The reaction mixture was diluted into 10 mL of C-18 buffer A, and the solution was loaded onto a preequilibrated C-18 SEP PAK cartridge and desalted as described above. The 5′-aminoethylphosphoramidate-derivatized oligomers were purified by electrophoresis on a 20% denaturing polyacrylamide gel. The oligomers were extracted from the gel by incubation of the gel slice with 1 mL of 0.1 M ammonium acetate, 20% aqueous acetoni-

500 Bioconjugate Chem., Vol. 15, No. 3, 2004

trile overnight at 37 °C. The oligomers were then desalted on C-18 SEP PAK cartridges. The 5′-aminoethylphosphoramidate derivatized oligomers (1.1 × 105 cpm) were each dissolved in 10 µL of RPMI medium supplemented with 10% fetal calf serum and incubated at 37 °C. One microliter aliquots were removed at various times, diluted into 4 µL of gel loading buffer, and analyzed on a 20% denaturing polyacrylamide gel. Products were visualized by phosphorimaging the wet gel. Percent intact oligonucleotide for each timepoint was quantitated using Image Quant 5.2 (Molecular Dynamics). Oligofectamine Uptake of Fluorescent Oligonucleotides in L929 Fibroblasts. Mouse L929 fibroblast cells were plated at a density of 55 000 cells per 1.9 cm diameter slide chamber 24 h prior to the experiment. For each experiment, 1 µL of Oligofectamine (Invitrogen) was preincubated with 9 µL of serum-free Eagle’s minimum essential media (EMEM) for 10 min at room temperature. For oligonucleotide 3-1mp-F, 0.1 nmol was resuspended in 90 µL of serum-free EMEM, then mixed with 10 µL of preincubated Oligofectamine/EMEM and incubated at room temperature for 20 min. The cells were washed twice with 500 µL of serum-free EMEM and incubated with a solution that contained 400 µL of serum-free EMEM and 100 µL of the preincubated oligonucleotide/ Oligofectamine mixture for 4 h in a 37 °C humidified CO2 incubator. Prior to viewing, the live cells were washed twice with 1 mL of Hanks Balanced Salt Solution (HBSS), and coverslips were mounted with HBSS. The live cells were viewed on an inverted Ziess fluorescence microscope. Phase contrast and green fluorescence images of the cells treated with fluorescent oligonucleotide with or without Oligofectamine were taken at both 40× and 100× magnification. Oligonucleotide Stability in Mouse L929 Fibroblasts. Oligonucleotides were phosphorylated by incubating 2.5 × 10-6 mmol oligonucleotide with 15 units T4 polynucleotide kinase in 15 µL of buffer that contained 250 µM γ-32P-ATP, (specific activity, 10 Ci/mmol), 50 mM Tris, pH 7.6, 10 mM magnesium chloride, and 10 mM mercaptoethanol overnight at 37 °C. The labeled oligomers were purified by electrophoresis on a 20% denaturing polyacrylamide gel and desalted as described above. The L929 cells were plated at a density of 70 000 cells/ well in 12-well tissue culture plates 24 h prior to the experiment. For each timepoint, 4 µL of Oligofectamine was incubated with 6 µL of serum-free EMEM for 10 min at room temperature. For each timepoint, 0.25 nmol of oligonucleotide was resuspended in 90 µL of serum-free EMEM and mixed with 10 µL of preincubated Oligofectamine/EMEM and incubated for 20 min at room temperature. The cells were washed twice with 500 µL of serum-free EMEM and incubated with a solution containing 400 µL of serum-free EMEM and 100 µL of the preincubated oligonucleotide/Oligofectamine mixture for 4 h in a 37 °C humidified CO2 incubator. After 4 h of incubation, all cells were washed twice with 500 µL of serum-free EMEM. At 4 h and beyond, the cells for the 8 and 24 h timepoints were incubated in 1 mL of EMEM containing 10% fetal calf serum. The cells were again washed twice with 500 µL of serum-free EMEM, then harvested by brief incubation with 500 µL of 50% aqueous acetonitrile, collected by scraping with a pipet tip, and stored at -20 °C until all timepoints were taken. The harvested cells in 50% acetonitrile were then incubated at -80 °C for 1 h to improve lysis. The majority of the cell debris was pelleted by centrifugation in a tabletop microcentrifuge at maximum speed for 3 s. The super-

Prater and Miller

natants were removed and quantitated by scintillation counting. Equivalent counts (about 550 cpm/lane) of each oligonucleotide timepoint were run on a 20% denaturing polyacrylamide gel. Gel-purified, 32P-labeled oligonucleotide stocks (4500 cpm/lane) were run as T0 controls. Products were visualized by phosphorimaging the dried gels overnight. Oligonucleotide Conjugation to Tat Peptide via 2, 2′-Dithiodipyridine. Cystamine dihydrochloride was exchanged on Amberlite quaternary amine resin (Fisher). Amberlite resin (5 g) was prepared in 20 mL of 0.5 N sodium hydroxide, poured into a 1.25 cm diameter glass column, and then equilibrated with 30 mL of 0.5 N sodium hydroxide, 50 mL of 0.1 N sodium hydroxide, and 200 mL of water. Cystamine dihydrochloride (3 mmol, 675 mg) was dissolved in deionized water and passed over the equilibrated Amberlite resin. Free cystamine was collected, evaporated, and stored at 4 °C. Controlled-pore glass (CPG) support-bound dT10 (Scheme 1, 1.1) was reacted with 81.7 mg (0.5 mmol) of carbonyldiimidazole in 1 mL of anhydrous acetonitrile for 2 h at room temperature in an ABI synthesis column. The support was washed with 10 mL of anhydrous acetonitrile and reacted with 3.4 mg of free cystamine in 200 µL of anhydrous acetonitrile at room-temperature overnight in a small Reactivial. The support was transferred back to the synthesis column and washed with 10 mL of anhydrous acetonitrile and dried on a vacuum line. The disulfide bond of the cystamine derivative was reduced by passing 1 mL of 100 mM dithiothreitol (DTT) in 50% aqueous acetonitrile through the column for 2 h at room temperature. The support was washed with 10 mL of 50% aqueous acetonitrile, followed by 10 mL of acetonitrile and dried. The support was transferred to a small Reactivial and incubated at 37 °C for 3 days in 100 µL of 0.05 M sodium phosphate buffer containing 50% acetonitrile and saturated with 6 mg of 2,2′-dithiodipyridine (Fluka). The support was then washed with 10 mL of 50% aqueous acetonitrile followed by 10 mL of acetonitrile and dried. The pyridyl disulfide-derivatized dT10 (Scheme 1, 1.5) was cleaved from the support by treatment with 0.4 mL of concentrated ammonium hydroxide for 2 h at room temperature. Full-length 1.6 was purified by C-18 HPLC. Each step of the derivatization was monitored by deprotecting a small amount of the support in 100 µL of concentrated ammonium hydroxide for 1 h. The oligonucleotide derivatizations were confirmed by C-18 HPLC and by MALDI-TOF mass spectrometry: 1.3 (m/z: calcd 3147.5, found 3146.8); 1.4 (m/z: calcd 3072.4, found 3072.2); 1.6 (m/z: calcd 3181.4, found 3182.6). Purified 1.6 (0.2 A260 units) was reacted with a 2-fold molar excess of Tat peptide in 5 µL of 50% aqueous acetonitrile overnight at room temperature. Conjugation of the oligonucleotide with Tat resulted in a substantial change in the mobility of the oligonucleotide when the reaction mixture was analyzed on a 20% denaturing polyacrylamide gel as detected by UV shadowing. The presence of the Tat-oligonucleotide conjugate in the reaction mixture was further confirmed by MALDI-TOF mass spectrometry: 1.7 (m/z:calcd 4891.5, found 4890.1). The above protocol was used for the conjugation of oligonucleotide 3-2mp-F with the Tat peptide except the reaction time in DTT was decreased to 30 min. Also, the majority of the pyridyl disulfide oligonucleotide 3-2mp-F was released from the CPG support during the pyridyl disulfide reaction and was recovered from solution. The pyridyl disulfide derivatized oligonucleotide, 3-2mp-F, was deprotected with ethylenediamine as described above for 2′-O-methyl methylphosphonate-containing oligonu-


Figure 1. General structure of an oligo- 2′-O-methylribonucleotide containing a single methylphosphonate linkage at the 3′ end. Table 1. Oligonucleotide Sequencesa oligomer

sequence

1 1-2mp 2 2-1mp 3 3-1mp 3-1mp-F 3-2mp 3-2mp-F 4 4-F

5′ mr-CpUpCpCpCpApGpGpCpUpCpApGpApU 3′ 5′ mr-CpUpCpCpCpApGpGpCpUpCpApGpApU 3′ 5′ mr-CpCpGpUpCpApGpCpGpUpCpApUpUpGpA 3′ 5′ mr-CpCpGpUpCpApGpCpGpUpCpApUpUpGpA 3′ 5′ mr-UpCpApUpUpGpApCpGpCpUpGpCpGpC 3′ 5′ mr-UpCpApUpUpGpApCpGpCpUpGpCpGpC 3′ 5′ mr-FpUpCpApUpUpGpApCpGpCpUpGpCpGpC 3′ 5′ mr-UpCpApUpUpGpApCpGpCpUpGpCpGpC 3′ 5′ mr-T*pUpCpApUpUpGpApCpGpCpUpGpCpGpC 3′ 5′ mr-CpGpUpCpA 3′ 5′ mr-FpCpGpUpCpA 3′

a The notations “mr-” and “d-“ denote 2′-O-methyl and deoxy sugars, respectively. Methylphosphonate linkages are denoted by “p” within the sequence, and the “#mp” suffix on the oligo name. T*p is 5-fluorescein derivative of deoxythymidine, whose structure is shown in Scheme 1, and Fp is a derivative of 6-carboxyfluorescein, whose structure is shown in Scheme 2.

cleotides, purified by C-18 HPLC, and reacted with the Tat peptide as above. The presence of Tat-conjugated 3-2mp-F was confirmed by MALDI-TOF mass spectrometry: (m/z: calcd 7670.2, found 7674.6). Oligonucleotide Conjugation to Tat Peptide via 4-(2-Aminooxyethoxy)-2-(ethylureido)quinoline. Tat peptide (0.5 mg) was reacted with 0.2 M bromoacetone in 50 µL of acetonitrile overnight at room temperature. The keto-Tat peptide product (Scheme 2, 2.4) was purified by C-18 HPLC using a 20 mL linear gradient of 2-35% acetonitrile in 0.1% TFA at a flow-rate of 1.0 mL/ min and analyzed by MALDI-TOF mass spectrometry: (m/z: calcd 1879.0, found 1878.3). The peptide was stored dry at -20 °C. Approximately 9.4 µg of keto-Tat peptide was reacted with 0.1 A260 unit of oligonucleotide AOQ-4-F (Scheme 2, 2.1) in the presence of 0.09 A260 unit of a dT21 in a total volume of 10 µL of 50% aqueous acetonitrile for 1 h at room temperature. Analysis of the reaction mixture by MALDI-TOF mass spectrometry confirmed the formation of the conjugate, Tat-4-F (m/z: calcd 4390.6, found 4392.0). RESULTS AND DISCUSSION

Oligonucleotides containing 2′-O-methylribonucleotides have been shown to have high binding affinity for RNA targets (2, 3, 7-11). Further modification with alternating methylphosphonate linkages has been shown to impart nuclease resistance to oligo 2′-O-methylribonucleotides while maintaining high binding affinity for comple-


mentary RNA (1, 2). Recent reports suggest that oligo2′-O-methylribonucleotides have some level of nuclease resistance (12-14) compared to oligodeoxyribonucleotides. Consequently incorporation of one or few methylphosphonate linkages into an oligo- 2′-O-methylribonucleotide already possessing partial nuclease resistance may be sufficient to impart nuclease resistance in vitro and in the cell. To test this possibility, we prepared oligo2′-O-methylribonucleotides that contain one (Figure 1) or two methylphosphonate linkages at the 3′-end of the molecule and studied their uptake by and stability within cells in culture. Oligo-2′-O-methylribonucleotides and Their Methylphosphonate Derivatives. The sequences of the three antisense oligo-2′-O-methylribonucleotides used in this study are shown in Table 1. Oligomer 1, a 15-mer that is complementary to nucleotides 21-35 (GenBank accession number NC_001802) of the TAR element of HIV-1 RNA, binds with high affinity to this site (1, 2). Oligomers 2, a 16-mer, and 3, a 15-mer, are complementary to nucleotides 7370-7385 and 7362-7385, respectively, of the Rev Response Element (RRE) of HIV-1 RNA (GenBank accession number NC_001802). Like the antiTAR oligomer, oligomers 2 and 3 bind with high affinity to their target RNA in vitro under essentially physiological conditions, that is at 37 °C in pH 7.0 buffer containing 0.1 M sodium chloride (unpublished data). The sequences of the methylphosphonate derivatives of these oligonucleotides are also shown in Table 1. Oligomers 1-2mp and 3-2mp contain two methylphosphonate linkages separated by a phosphodiester linkage at their 3′-end. Oligomers 2-1mp and 3-1mp contain a single 3′-terminal methylphosphonate linkage. Oligomers 1, 2 and 3 were synthesized using commercially available, protected 2′-O-methylribonucleoside3′-O-β-cyanoethyl-(N,N-diisopropyl)phosphoramidites. These oligomers were deprotected by treatment with concentrated ammonium hydroxide at 55 °C and were purified by strong anion exchange (SAX) HPLC. The methylphosphonate-derivatized oligomers were prepared using commercially available 2′-O-methylribonucleoside3′-O-methylphosphonamidites. To avoid cleavage of the methylphosphonate linkages during deprotection, the oligomers were treated briefly with concentrated ammonium hydroxide at room temperature followed by treatment with ethylenediamine at room temperature (2). These oligonucleotides were purified either by SAX HPLC or by preparative polyacrylamide gel electrophoresis. Stability of Oligo-2′-O-methylribonucleotides in Serum. The stabilities of the 5′-[32P]-labeled oligonucleotides in RPMI culture medium containing 10% fetal calf serum (FCS) was determined. To prevent possible loss of label due to serum phosphatase activity, the 5′terminal phosphate group was converted to its 2-aminoethyl phosphoramidate derivative by reaction with ethylenediamine in the presence of 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide (2). The labeled oligonucleotides were incubated in RPMI medium containing 10% FCS for up to 24 h at 37 °C. The reaction mixtures were then analyzed by polyacrylamide gel electrophoresis, and the gels were visualized by phosphorimaging. Figure 2, panel A, compares the stability of allphosphodiester oligonucleotide 1 with that of oligomer 1-2mp, which contains two alternating methylphosphonate linkages at it’s 3′ end. As shown in panel E, which shows the percentage of intact oligomer versus time, essentially all of 1-2mp remains intact after a 2 h incubation, whereas only 18% of intact oligomer 1 is


Prater and Miller

Figure 2. Stabilities of oligonucleotides in 10% fetal calf serum. 32P-labeled oligonucleotides were incubated in medium containing 10% serum for the indicated lengths of time, and the reaction mixtures were analyzed by polyacrylamide gel electrophoresis. The gels (A) 1 vs 1-2mp; (B) 2 vs 2-1mp; (C) 3 vs 3-1mp; (D) dT10 were visualized by phosphorimaging. (E) Quantitation of percent intact oligonucleotide at each timepoint: oligonucleotide 1 (b), 1-2mp (O), 2 (1), 2-1mp (3), 3 (9), 3-1mp (0), d-T10 ([).

observed. Even after an 18 h incubation, approximately 55% of 1-2mp remains intact. Oligomers 2-1mp and 3-1mp, each of which contains a single 3′-methylphosphonate linkage, display stabilities similar to that of 1-2mp (Figure 2B and C). Thus after a 2 or 4 h incubation, between 80% to 95% of these oligomers remain intact. Even after a 24 h incubation, between 30% and 60% of each oligomer is observed intact. As was the case for oligomer 1, the all-phosphodiester oligomers 2 and 3 were degraded approximately 80% after 2 h of incubation. However, consistent with previous findings, the rates of degradation of these all-phosphodiester oligo-2′-O-methylribonucleotides were significantly less than that of dT10, an all-phosphodiester oligodeoxyribonucleotide that was 80% degraded after 0.5 h of incubation (Figure 2D and E). These data are consistent with our previous results for the serum stability of a methylphosphonate-derivatized hairpin oligo-2′-O-methylribonucleotide (4) and with oligodeoxyribonucleotides having a 3′-terminal methylphosphonate linkage (15). The combined results show that methylphosphonate linkages located in the 3′-terminal portion of the oligomer can impart considerable protection against degradation by 3′-exonuclease activity found in mammalian sera. Furthermore, addition of a single methylphosphonate linkage at the 3′ end of oligonucleotides 2 and 3 is sufficient to significantly enhance the serum exonuclease resistance of the 2′-O-methyl sequences (Figure 2E) and addition of a second (alternat-

ing) methylphosphonate linkage to oligo 3-1mp did not further enhance the nuclease resistance of the oligomer (data not shown). In separate studies, we have found that the single methylphosphonate linkage in either oligomer 2-1mp or 3-1mp does not perturb binding of the oligomer with its RRE RNA target (unpublished data) Stability of Oligo-2′-O-methylribonucleotides in Mouse L929 Fibroblasts. We next examined the stability of the oligo-2′-O-methylribonucleotides and their methylphosphonate derivatives in mouse L929 fibroblasts. Cationic lipids are commonly used to transfect oligonucleotides into cells in culture (16, 17). Complexes of Oligofectamine, a cationic lipid, and 32P-labeled oligonucleotides 2, 2-1mp, 3, 3-1mp, or dT15 were prepared in serum-free medium. Mouse L929 cells were incubated with the complexes for 4 h at 37 °C after which the cells were washed with serum-free EMEM and incubated an additional 0, 4, or 20 h with fresh oligonucleotide-free medium. No morphological changes in the cells indicative of toxicity were observed even after 24 h of incubation. The cells were then washed and lysed, cell debris were removed by centrifugation, and the amounts of radioactivity from the oligonucleotides were measured by scintillation counting. The results are shown in Table 2. Introduction of a single methylphosphonate linkage reduces the net charge of the oligonucleotide by one, which might be expected to affect the uptake of oligonucleotides in the presence of cationic lipids. This appears to be the case for oligonucleotides 2 versus 2-1mp, as



Figure 3. Stabilities of oligonucleotides in mouse L929 cells in culture. Mouse L929 fibroblasts were incubated with Oligofectamine complexes of the 32P-labeled oligonucleotides for the indicated lengths of time. Lysates from the washed cells were analyzed by polyacrylamide gel electrophoresis, and the gels were visualized by phosphorimaging. Table 2. Oligonucleotide Recovery from L929 Cell Lysatesa oligonucleotide pmols recovered

2

2-1mp

3

3-1mp

dT15

4.61

1.14

11.8

11.8

0.335

a

pmol of oligonucleotide recovered after Oligofectamine delivery into L929 cells. Quantitation based on scintillation counts of cell lysates after 4 h of incubation.

introduction of a single methylphosphonate linkage results in a significant decrease in the uptake of the oligonucleotide/Oligofectamine complex as determined by oligonucleotide recovery after 4 h (Table 2). However, essentially no difference in uptake is seen for oligonucleotides 3 versus 3-1mp, which also differ by a single negative charge. These observations would suggest that in addition to overall charge, oligonucleotide sequence and chain length also contribute to cationic lipid-mediated uptake. Consistent with this idea is the observation that oligonucleotides 2-1mp and 3, which both have a net negative charge of -14 but differ in their size and sequence, show significantly different uptake when complexed with Oligofectamine. To determine the extent of degradation of each oligonucleotide, radioactivity from the cell lysates was analyzed by denaturing polyacrylamide gel electrophoresis. The phosphorimages of these gels are shown in Figure 3. Similar to the behavior of dT10 in the in vitro serum stability assay (Figure 2), the oligodeoxyribonucleotide dT15 was degraded rapidly to give a “ladder” of oligonucleotides each differing by one nucleotide unit. The decreasing intensities of the shorter oligonucleotides in this ladder suggest that degradation occurs in a stepwise manner starting at the 3′-end of the dT15. Such behavior would be consistent with the activity of a 3′-exonuclease. Only 40% of recovered dT15 oligonucleotide remains intact after 4 h incubation in L929 cells. Unlike the oligodeoxyribonucleotide, both the oligo-2′-O-methylribonucleotides 2 and 3, as well as their methylphosphonate derivatives 2-1mp and 3-1mp, were remarkably resistant to cellular nuclease degradation. Due to low oligonucleotide recovery of sequence 2-1mp after 24 h cell incubation, the corresponding lane in Figure 3 is underloaded with respect to adjacent lanes. As expected, the methylphosphonate-containing oligonucleotides 2-1mp and 3-1mp were recovered intact, with no evidence of degradation throughout the 24-h experiment Surprisingly however, similar results were seen with the all-phosphodiester oligomers 2 and 3. Despite the appearance of a minor n-1 band in the later timepoints of panels 2 and 3 in Figure 3, 99% of the recovered oligo-2′-O-methylri-

bonucleotide is full-length even after 24 h of incubation with L929 cells. This behavior, which is in contrast to that seen in the in vitro serum stability assay (Figure 2), suggests that the oligonucleotides may be protected from degradation by the cationic lipids and/or their exposure to exonuclease activity is limited within this particular intracellular environment. Cationic lipids generally deliver oligodeoxyribonucleotides and oligodeoxyribonucleotide phosphorothioates to the nucleus. The localization of the fluorescein derivative of methylphosphonate oligo-2′-O-methylribonucleotide 3-1mp was determined using fluorescence microscopy. The fluorescein derivative of 3-1mp was synthesized using a commercially available fluorescein phosphoramidite and after deprotection was purified by SAX HPLC. The resulting fluorescein-derivatized oligomer, 3-1mpF, was complexed with Oligofectamine and incubated with mouse L929 fibroblasts for 4 h at 37 °C. To avoid potential artifacts due to fixing (18), the live cells were then washed and viewed by fluorescence microscopy as previously described (19). Panels A and B of Figure 4 show a representative field of cells at 40× magnification, with approximately 70% (19/26) of the live cells exhibiting fluorescence, and 40% (11/26) of the cells exhibiting nuclear fluorescence. As shown in Figure 4C, the most intense fluorescence was observed in the nucleus, with the exception of several punctate regions that may represent endosomal entrapment of the oligonucleotide/ Oligofectamine complex. Additionally, weak fluorescence was observed in the cytoplasm. These results are similar to those seen for cationic lipid complexes of fluorescently derivatized all phosphodiester oligo-2′-O-methylribonucleotide 4-F (data not shown). Primary localization of the oligo-2′-O-methylribonucleotide/Oligofectamine complexes in the nuclear compartment may explain their impressive stability in L929 cells versus the in vitro serum assay. Tat Peptide-Oligonucleotide Conjugates. The experiments described above show that Oligofectamine can be used to deliver oligo-2′-O-methylribonucleotides and their methylphosphonate derivatives to the nuclei of mouse fibroblasts. Recent research with “protein transduction domains” (PTDs) has led to the discovery of several peptides that are reported to translocate conjugated molecules, including oligonucleotides, through the plasma membrane. This group of so-called cellpenetrating peptides contains highly basic regions from some well-known proteins including Antennapedia, VP22 herpes virus protein, and HIV-Tat (20). Although the mechanism by which cell-penetrating peptides deliver their cargo to the nucleus is not well understood and is the subject of some debate (18, 21-25), a number of studies have reported enhanced antisense activity of


Prater and Miller

Figure 4. Fluorescence photomicrograph of mouse L929 fibroblasts treated with a complex of Oligofectamine and oligonucleotide 3-1mp-F. Panels A and B show a representative field of cells at 40× magnification in fluorescence and phase-contrast modes, respectively. Panel C, single cell, 100× magnification. Scheme 1. Tat-Oligonucleotide Conjugate Formation via Dithiodipyridinea

a Reagents: (i) carbonyldiimidazole; (ii) cystamine; (iii) dithiothreitol; (iv) dithiodipyridine; (v) deprotection and purification (see methods); (vi) Tat-SH.

oligonucleotides (26-29) and peptide nucleic acids (3032) when covalently conjugated to cell-penetrating peptides. To explore the potential of this alternative delivery strategy, we attempted to prepare Tat derivatives of fluorescently labeled oligo-2′-O-methylribonucleotides and study their uptake by mouse L929 fibroblasts in culture. Two approaches were used to synthesize Tat-conjugated oligonucleotides. The first, which is outlined in Scheme 1, employed disulfide-coupling chemistry to attach Tat to the oligonucleotide. We first attempted to carry out the derivatization and coupling chemistry on CPG-bound dT10. The potential advantage of this approach is the oligonucleotide would remain bound to the CPG throughout the entire conjugation process, allowing excess reagents to be washed away and thus requiring minimal purification steps. Support-bound dT10 (Scheme 1, 1.1) was reacted with carbonyldiimidazole to give the 5-imidazole carbamate intermediate, 1.2, which was then

converted to the 5′-cystaminyl derivative, 1.3. The disulfide bond of 1.3 was reduced with dithiothreitol, and the resulting free sulfhydryl group was reacted with 2,2′dithiodipyridine to give 5′-pyridyl disulfide derivatized oligonucleotide 1.5. The progress of each derivatization step was monitored by deprotecting a small portion of the support and analyzing the reaction mixture by C-18 reversed phase HPLC. The compositions of the intermediates were also confirmed by MALDI-TOF mass spectrometry. We encountered problems when we attempted to couple the C-terminal cysteine sulfhydryl group of Tat peptide (GRKKRRQRRRPPQC), to the oligonucleotide by exchange with the pyridylsulfide group. It appeared that the resulting conjugate strongly adhered to the CPG after ammonium hydroxide cleavage because no oligonucleotide could be recovered from the support. To circumvent this problem, we removed the 5′-pyridyl disulfide-deriva-



Scheme 2. Tat-Oligonucleotide Conjugate Formation via 4-(2-Aminooxyethoxy-2-(ethylureido)quinolinea

a

Reagents: (i) bromoacetone; (ii) AOQ phosphoramidite; (iii) deprotection and purification (see methods); (iv) keto-Tat (2.4).

tized dT10, 1.5, from the support and purified it by C-18 reversed phase HPLC. The purified oligonucleotide, 1.6, was then reacted with an excess of Tat peptide. Examination of the reaction mixture by polyacrylamide gel electrophoresis showed the presence of a new compound whose reduced electrophoretic mobility was consistent with formation the conjugate. Analysis of the reaction mixture by MALDI-TOF mass spectrometry confirmed the presence of the Tat-dT10 conjugate, 1.7, and suggested that all of the dT10 had been converted to conjugate. The same reaction protocol was applied to the fluorescein conjugate of oligomer 3-2mp, 3-2mp-F. This oligonucleotide was prepared using a commercially available fluorescein-dT phosphoramidite. The support-bound oligomer, 1.1, was converted to its 5′-pyridyl disulfide derivative, 1.6, as described above for the preparation of the dT10 derivative. Reaction of purified oligonucleotide with the Tat peptide resulted in almost immediate formation of a sticky orange precipitate. Similar behavior has been reported for the reaction of Tat peptide with the pyridyl disulfide derivatives of rhodamine- and fluorescein-conjugated oligonucleotides (33, 34). Analysis of the reaction mixture by MALDI-TOF mass spectrometry confirmed the presence of the Tat-3-2mp-F conjugate. Precipitation did not occur when Tat peptide was added to fluorescein derivatized oligomer that lacked a 5′-pyridyl disulfide group, suggesting that precipitation was a property of the covalent Tat-oligonucleotide conjugate. This aggregation phenomenon proved to be an insurmountable problem in our hands and essentially prevented the purification of the conjugate in any useful amounts by C-4, C-8, or C18 reversed phase HPLC, strong anion exchange HPLC, gel filtration, diethylami-

noethyl (DEAE), or carboxymethyl (CM) Sephadex. When the fluorescinated Tat-oligonucleotide conjugate was applied to DEAE or CM Sephadex loaded in a glass column, most of the conjugate appeared as a visible orange band, which could not be eluted from the column. In addition to this aggregation problem, we found that the Tat-3-2mp-F conjugate adhered to plastic and glass tubes, behavior that has also been observed with Tat conjugates of peptides (35). In a second approach, we used oxime formation to conjugate Tat to a fluorescein derivative of penta-2′-Omethylribonucleotide 4. Oligomer 4 was synthesized on controlled pore glass and derivatized with 6-carboxyfluorescein phosphoramidite to give support-bound oligomer 2.1 as shown in Scheme 2. The hydroxyl group of 2.1 was reacted with 4-(2-aminooxyethoxy-2-(ethylureido)quinoline phosphoramidite (5) to give 4-(2-aminooxyethoxy-2(ethylureido)quinoline (AOQ) derivative 2.2. The AOQderivatized oligomer was deprotected and purified by C-18 reversed phase HPLC to give oligomer 2.3. Conjugation of 2.3 with keto-Tat derivative 2.4, formed by reaction of Tat peptide with bromoacetone (5), gave Tat conjugate 2.5, which precipitated from the reaction mixture. We found that this precipitation could be prevented by running the reaction in the presence of an excess of dT21. Under these conditions, the reaction solution remained clear. The extent of the reaction could be determined by following the disappearance of AOQoligomer 2.3 on C-18 reversed phase HPLC. Based on this criterion, the reaction appeared to go to completion. However, we were unable to observe Tat conjugate 2.5 on the HPLC column, although we could detect the presence of the conjugate in the reaction mixture by MALDI-TOF mass spectrometry.


It is unclear why the Tat conjugates of the fluoresceinderivatized oligomers are insoluble. As described above, simply mixing Tat peptide with the oligonucleotide does not result in precipitation. Rather it appears that Tat must be linked covalently with the oligonucleotide for precipitation to occur. Possibly formation of a covalent conjugate between the negatively charged oligomer and the positively charged Tat peptide promotes intramolecular electrostatic interactions between the oligomer and Tat peptide that lead to a decreased solubility of the conjugate (36). Consistent with this idea is the observation that addition of dT21 prevented precipitation of the Tat-4-F during the conjugation reaction. Under these conditions, the negatively charged dT21 could interact with the positively charged Tat peptide to help prevent precipitation of the conjugate. However, even in the presence dT21 we found that Tat-4-F was strongly absorbed to glass surfaces (data not shown), and this absorption served as an impediment to further studying the uptake of these oligonucleotides in cell culture. CONCLUSIONS

Our studies show that 3′-methylphosphonate-modified oligo-2′-O-methylribonucleotides are stable to the 3′exonuclease activity found in mammalian serum. These oligonucleotides may be transfected into cells in culture by complex formation with cationic lipids, which deliver the oligonucleotides to the cell nucleus. Oligonucleotides delivered in this manner can be recovered intact from the cells suggesting that they are stable in the intracellular environment. These properties, along with their ability to bind with high affinity to complementary RNA targets, suggest that 3′-methylphosphonate-modified oligo2′-O-methylribonucleotides may be suitable for use as antisense agents in cell culture. Our experience with the Tat-conjugated oligonucleotides suggests that they are prone to aggregation, which makes purification difficult, and that they have high affinity for glass and plastic surfaces. These properties and the recent reports questioning the mode of uptake of Tat-conjugated molecules (18, 21-25) suggests that caution should be used when designing Tat-oligonucleotides for uptake studies. ACKNOWLEDGMENT

The authors thank Dr. Michael Matunis for the use of his fluorescence microscope, and members of the Matunis laboratory for their valuable assistance. We thank Dr. Tomoko Hamma for providing us with oligonucleotide 1, and for her assistance in the preparation of the AOQ phosphoramidite. This research was supported by a grant from the National Institutes of Health, GM057140. LITERATURE CITED (1) Miller, P. S., and Hamma, T. (1999) Studies on anti-HIV oligonucleotides that contain alternating methylphosphonate/ phosphodiester linkages. Antisense Nucleic Acid Drug Dev. 9, 367-370. (2) Hamma, T., and Miller, P. S. (1999) Syntheses of alternating oligo-2′-O-methylribonucleoside methylphosphonates and their interactions with HIV TAR RNA. Biochemistry 38, 1533315342. (3) Miller, P. S., Cassidy, R. A., Hamma, T., and Kondo, N. S. (2000) Studies on anti-human immunodeficiency virus oligonucleotides that have alternating methylphosphonate/phosphodiester linkages. Pharmacol. Ther. 85, 159-163. (4) Hamma, T., and Miller, P. S. (2003) Interactions of hairpin oligo-2′-O-methylribonucleotides containing methylphosphonate linkages with HIV TAR RNA. Antisense Nucleic Acid Drug Dev. 13, 19-30.

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