Properties of Double-Stranded Oligonucleotides Modified with

Jul 30, 2010 - Brian M. Laing , Rudolph L. Juliano. ChemBioChem ... Agnieszka B. Olejniczak , Ryszard Kierzek , Eric Wickstrom , Zbigniew J. Lesnikows...
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Bioconjugate Chem. 2010, 21, 1537–1544

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Properties of Double-Stranded Oligonucleotides Modified with Lipophilic Substituents Brian M. Laing,† Lisa Barrow-Laing,‡ Maureen Harrington,§ Eric C. Long,| and Donald E. Bergstrom*,† Department of Medicinal Chemistry and Molecular Pharmacology and Bindley Bioscience Center, Purdue University, West Lafayette, Indiana 47907, Department of Microbiology and Immunology, and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, Department of Chemistry and Chemical Biology, Purdue School of Science, Indiana University-Purdue University Indianapolis (IUPUI), Indianapolis, Indiana 46202. Received April 25, 2010; Revised Manuscript Received June 1, 2010

We have synthesized a series of short, self-complementary oligonucleotide sequences modified at their 5′- and/or 3′- termini with a lipophilic dodecane (C12); these systems serve as models to assess the biophysical properties of double-stranded DNA (dsDNA) equipped with potentially stabilizing lipophilic substituents. Addition of C12 to the 5′-termini of self-complementary 10 nucleotide sequences increased their duplex melting temperatures (Tm) by ∼4-8 °C over their corresponding unmodified sequences. C12 functionalities added to both the 3′- and 5′-termini increased Tm values by ∼10-12 °C. The observed increases in Tm correlated with greater duplex stabilities as determined by the free energy values (∆G) derived from Tm plots. There is a greater degree of stabilization when C12 is positioned with a C · G base pair at the termini, and the stabilizing effect of lipophilic groups far exceeds the effect seen in adding an additional base pair to both ends of DNA. Stable, short dsDNA sequences are of potential interest in the development of transcription factor decoy oligonucleotides as possible therapeutic agents and/or biological tools. These results suggest that the stability of short dsDNA sequences are improved by lipophilic substituents and can be used as the basis for the design of dsDNAs with improved biological stabilities and function under physiological conditions.

INTRODUCTION The conjugation of lipophilic groups to oligonucleotides (ONs1) is closely linked to their development as antisense-based therapeutics (1-4). Conjugation of lipophilic groups to the 5′and/or 3′-termini of ONs have been shown to significantly improve cell uptake and antisense activity (3, 5-9) while also providing resistance to nucleases (10-12). The hybridization properties of ON-lipid conjugates have been studied and reported to result in stabilization (6, 13), destabilization (5, 6), or no effect on duplex stability (6, 14-16); however, it is important to note that these studies were done in the context of the antisense ONs and that the effects of strategic placement of the lipophilic moieties on the hybridization of dsDNA were not considered in most cases. To optimize the type and placement of lipophilic groups on oligonucleotides in order to facilitate cell uptake and intracellular trafficking, it is important to consider how natural biomolecules are tagged by lipophilic groups. Proteins are often conjugated to myristoyl (C14), palmitoyl (C16), farnesyl (C15), and geranylgeranyl (C20) groups (17, 18) that function as anchors for reversible association with cell membranes. Dual lipid * To whom correspondence should be addressed. Department of Medicinal Chemistry and Molecular Pharmacology, Birck Nanotechnology Center, Purdue University, 1205 W. State St., West Lafayette, IN 47907-2057. Tel: (765) 494-6275. Fax: (765) 494-1414. E-mail: [email protected]. † Purdue University. ‡ Department of Microbiology and Immunology, Indiana University School of Medicine. § Department of Biochemistry and Molecular Biology, Indiana University School of Medicine. | Indiana University-Purdue University Indianapolis (IUPUI). 1 Abbreviations: ON, oligonucleotides, dsDNA, double-stranded DNA, Tm, melting temperature.

Figure 1. Design of end-capped dsDNA decoy sequences with C12 at the 5′- and 3′-termini for enhanced stability and cell uptake. Fluorescent probes may be attached to either the 5′-end (red circle) or to an internal base (green circle).

modification often occurs on two adjacent amino acids; for example, a terminal glycine may be N-acylated with myristic acid and an adjacent cysteine S-acylated with palmitic acid. This dual modification appears to facilitate association with lipid rafts (19). In order to mimic this cell association behavior, we envisioned the development of dsDNA decoys comprising a single sequence containing self-complementary segments linked by a spacer and terminated at the 3′- and 5′-ends by a myristic acid mimic (Figure 1). The dodecane hydrocarbon chain (C12) was chosen because it almost exactly spans the diameter of the blunt end of a DNA duplex and contains nearly as many methylene groups as myristic acid (11 vs 12, respectively). On self-assembly in aqueous solution, the modified oligonucleotide

10.1021/bc100201n  2010 American Chemical Society Published on Web 07/30/2010

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Figure 2. Model system for assessing the biophysical properties of dsDNA conjugated to lipophilic substituents at the 5′- and 3′-termini. (A) Association of self-complementary 10-mer sequence with C12 conjugated to the 5′-terminus. (B) Association of self-complementary 10-mer sequence with C12 conjugated 3′-terminus. (C) Association of self-complementary 10-mer sequence with C12 conjugated to both the 5′- and 3′-termini.

would assemble in such a way that the two terminal C12 groups could fold across the top of the helix. However, the C12 groups could also fold outward to allow the oligonucleotide to anchor into a cell membrane (Figure 1). Membrane integration is a critical step in localization and the eventual uptake of the ON by the cell. Just as acylation by lipophilic hydrocarbons can stabilize protein secondary structure (20), it seemed likely that terminal C12 groups could stabilize DNA secondary structure. In this study, we assessed the effect of terminal lipophilic (C12) groups on the hybridization of double stranded DNA. We used short (10-mer) self-complementary sequences to which we attached a 12-carbon alkyl group to the 5′- and 3′-termini as a model system for assessing the relative effect of lipophilic groups on duplex thermal stability (Figure 2). With this simple model, we were able to obtain thermal melting profiles from which we extracted the melting temperature (Tm) and the thermodynamic parameters ∆H, ∆S, and ∆G. We hypothesized that lipophilic groups attached to the 3′- and 5′-ends of dsONs will selfassociate in aqueous solution, thus conferring increased thermal stability. The models utilized in this study are simple alkyl groups in contrast to more lipophilic moieties (e.g., cholesterol) or highly engineered cross-linking molecules used in previous studies (21). The results presented here show that there is significant stabilization on addition of simple lipophilic alkyl groups to the termini of dsDNA.

EXPERIMENTAL PROCEDURES General Information. Reagents for organic synthesis were purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI) and were, unless otherwise stated, used as obtained. Anhydrous solvents were distilled from appropriate drying agents or

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purchased in Sure-Seal containers. Aminopropyl CPG was purchased from Biosearch Technologies, Inc. (Novato, CA). Standard base monomers, DyLight DY647 phosphoramidite, and reagents for oligonucleotide synthesis were purchased from Glen Research Corp. (Sterling, VA). Thin layer chromatography was performed using Sigma-Aldrich silica gel on glass 60F-254 TLC plates. Purification of compounds by silica gel chromatography was performed using Aldrich 230-400 mesh 60 Å silica gel. Nuclear magnetic resonance spectra were obtained using 300 or 500 MHz Bruker spectrometers. 1H and 13C spectra were referenced to TMS, while 31P spectra were referenced to an 85% phosphoric acid external standard. C12 Monomer Synthesis. Dodecyl 2-Cyanoethyl N,N Diisopropylphosphoramidite (2). 1-Dodecanol (0.22 mL, 1 mmol) was dissolved in 10 mL of anhydrous dichloromethane in a dry 25 mL two-necked flask. Diisopropylethylamine (0.63 mL, 3.6 mmol) was added while stirring under argon followed by 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.27 mL, 1.2 mmol). The reaction was allowed to proceed for 1 h at room temperature and then quenched by the addition of four drops of anhydrous methanol and 20 mL of CH2Cl2. The reaction mixture was transferred to a dry 125 mL separatory funnel and washed quickly with 5% NaHCO3 (10 mL × 2) and brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (4:1 EtOAc/hexane, 1% Et3N) to give 2 as a yellow oil (0.34 g, 88%). 1 H NMR (300 mHz, CDCl3, δ, ppm): 3.83 (t, 2H, J ) 6 Hz), 3.65-3.58 (m, 4H), 2.639 (t, 2H, J ) 6 Hz), 1.60 (m, 2H), 1.33-1.20 (m, 18H), 1.19 (dd, 12H), 0.88 (t, 3H, J ) 6 Hz). 31 P NMR (100.8 MHz, CDCl3, δ, ppm): 148.23 (referenced to external 85% H3PO4). HR (ESI)-MS: calculated (M + H)+ 387.3140; found, 387.3137. 12-(4,4′-Dimethoxytrityloxy)-1-dodecanol (4). Dodecane-1,12diol (5.0 g, 29.5 mmol) was coevaporated with dry pyridine (3 × 20 mL) and dissolved in dry pyridine (30 mL) with stirring under argon. Dimethoxytrityl chloride (2.0 g, 5.9 mmol) was dissolved in dry pyridine (20 mL) and the solution added dropwise with constant stirring. The reaction was allowed to proceed overnight and quenched with methanol (1 mL). Pyridine was removed under vacuum, the mixture dissolved in dichloromethane (200 mL), and filtered. The filtrate was washed with 5% NaHCO3 (200 mL × 2) followed by brine (100 mL) and dried over anhydrous sodium sulfate. The solution was concentrated under vacuum and the crude product purified by silica gel chromatography (1:1 ether/hexanes, 1% Et3N). The appropriate fractions were combined, evaporated, and dried in vacuum to give 4 as a pale yellow foam (0.9 g, 30%), Rf 0.18 (1:1 ether/hexanes, 1% Et3N). 1H NMR (500 MHz, CDCl3, δ, ppm): 7.43 (d, 2H, J ) 5 Hz), 7.31-7.33 (m, 4H), 7.27-7.29 (m, 2H), 7.19 (t, 1H, J ) 5 Hz), 6.80-6.83 (m, 4H), 3.79 (s, 6H), 3.63(q, 2H, J ) 5.5 Hz), 3.02 (t, 2H, J ) 6.5 Hz), 1.55-1.61 (m, 4H), 1.21-1.36 (m, 16H). 13C NMR (125.77 MHz, CDCl3, δ, ppm): 158.26, 145.47, 136.79, 130.02, 129.13, 128.21, 127.85, 127.76, 127.66, 126.52, 112.93, 85.59, 65.86, 63.49, 63.09, 55.19, 32.82, 30.09, 29.61, 29.58, 29.53, 29.43, 26.31, 25.74, 15.28. HR (ESI)-MS: calculated (M + Na)+ 527.3137; found, 527.3134. 12-(4,4′-Dimethoxytrityloxy)dodecyl 2-Cyanoethyl-N,N-diisopropylphosphoramidite (5). C12-DMT (0.66 g, 1.31 mmol) was coevaporated twice with anhydrous CH2Cl2 (10 mL) and then dissolved in CH2Cl2 (10 mL). DIEA (0.68 mL, 3.92 mmol) was added with stirring under argon followed by the addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.44 mL, 1.96 mmol). The mixture was stirred at room temperature for 2 h and monitored by TLC (1:1 ether/hexane, 1% Et3N). The reaction was quenched with 4 drops of anhydrous methanol and

Lipid Modified Double-Stranded Oligonucleotides

the subsequent addition of 20 mL of CH2Cl2. The reaction mixture was transferred to a dry 125 mL separatory funnel and washed quickly with 5% NaHCO3 (10 mL × 2) and brine (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (1:1 ether/hexane, 1% Et3N). The appropriate fractions were combined, evaporated, and dried in vacuum to give the product as a pale yellow oil (0.73 g, 79%), Rf 0.48 (1:1 ether/hexane 1% Et3N). 1H NMR (500 MHz, CDCl3, δ, ppm): 7.43 (d, 2H, J ) 5 Hz), 7.31-7.33 (m, 4H), 7.27-7.29 (m, 2H), 7.19 (t, 1H, J ) 5 Hz), 6.80-6.83 (m, 4H), 3.80-3.88 (m, 2H), 3.79 (s, 6H), 3.55-3.65(m, 4H), 3.022 (t, 2H, J ) 6.5 Hz), 2.63 (t, 2H, J ) 6.5 Hz), 1.6 (p, 4H, J ) 7 Hz), 1.55 (s, 1H), 1.21-1.35 (m, 16H), 1.16-1.19 (m, 11 H). 13C NMR (125.77 MHz, CDCl3, δ, ppm): 158.27, 145.48, 136.79, 130.02, 128.21, 127.66, 126.52, 112.93, 99.98, 85.59, 63.82, 63.51, 58.38, 58.23, 55.19, 43.02, 42.92, 31.20, 30.12, 29.62, 29.36, 26.34, 25.95, 24.68, 24.63, 24.58, 24.52, 20.38, 20.32. 31P NMR (202.45 MHz, CDCl3, δ, ppm): 147.81 (referenced to external 85% H3PO4). HR (ESI)-MS: calculated (M + H)+, 705.4396; found, 705.4403. Preparation of C12 CPG. 3-N-(1,3,4-Trimellityl-3,4anhydride)aminopropyl-CPG (7). Aminopropyl-CPG (5 g) was placed in a 125 mL Erlenmeyer flask and closed with a stopper. A solution of trimellitic anhydride chloride (1 g) in a mixture of pyridine (10 mL) and methylene chloride (40 mL) was then added. The mixture was placed on a solid phase shaker for 2 h. The CPG was poured in a sintered glass funnel and washed with methylene chloride (3 × 50 mL) and acetonitrile (3 × 50 mL). The support was dried in a vacuum desiccator and tested for the presence of free amino groups with a Kaiser test. The procedure was repeated until the Kaiser test was found to be negative. 1-Dimethoxytrityl-O-dihydroxydodecyl-3-O-(4′- or 5′-trimellityl)1′-N-trimellityl)aminopropyl-CPG (9). Compound 7 (2 g) was placed in a 125 mL Erlenmeyer flask. 1,12-Dodecanediol (2.6 g) was dissolved in 10 mL of DMF and added to the flask followed by N-methylimidazole (0.5 mL). The flask was placed on a solid phase shaker for 18 h. The support was washed and dried as above. Dimethoxytrityl chloride (1 g) in 100 mL of pyridine was added to the flask and placed on a shaker for 16 h. The support was washed and dried as described above. The dimethoxytrityl (DMT) loading, as determined by colorimetric DMT loading assay, was 15 µmol/g. Molecular Modeling. Molecular modeling analyses were carried out using Sybyl (Tripos) (35) installed on a Silicon Graphics IRIS 4D/120GTX workstation. B-DNAs were generated using the biopolymer build module. The straight chain alcohol C12H25OH and the straight chain diol HOC12H24OH were sketched using the sketch molecules tool in Sybyl. The sketched molecules were then minimized using the Kollman force field. Minimization followed the Powell conjugate gradient method for up to 10000 iterations until the gradient change in energy was 0.01 kcal/mol or less. The molecule was solvated with explicit water molecules, and aggregates were defined in the structure such that the minimization would only involve the terminal base pairs adjacent to the hydrophobic groups. Oligonucleotide Synthesis. Oligonucleotides were synthesized on an Applied Biosystems 392 DNA/RNA synthesizer using standard phosphoramidite chemistry. The syntheses were initiated on columns containing controlled pore glass support loaded with the required 3′ nucleoside phosphoramidite or 3′ C12 for the first base in the synthesis. Syntheses were carried out on a 1 µmole scale, and the standard coupling time (25 s) was used for the standard nucleoside phosphoramidites and an extended coupling time of 300 s (2×) used for the internal C12 phosphoramidites (for cell uptake studies - see Supporting

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Information), 5′ C12 phosphoramidites, and 5′ -DyLight 647 phosphoramidite. For sequences with fluorescent groups, the ultramild base protected nucleoside phosphoramidite monomers and phenoxyacetyl protected adenine (pac-dA), 4-isopropylphenoxyacetyl protected guanine (ipr-pac-dG), and acetyl protected cytosine (ac-dC) were used to facilitate shorter deprotection times with ammonium hydroxide. Unmodified sequences were synthesized with DMT-on for purification by either reversed phase HPLC or cartridge purification (Glen-Pak or Polypack cartridges, Glen Research, Sterling VA). Both purification methods depend on the hydrophobicity imparted by the DMT group to the oligonucleotide, thus significantly increasing the retention time on a C18 HPLC column or cartridge. C12 modified sequences were synthesized with DMT-off since the C12 modification imparts a similar hydrophobic effect on the oligonucleotides, facilitating easy purification on a C18 reversed phase HPLC column. Some longer double stranded sequences were found to be too hydrophobic for purification on a C18 column and were purified with a less hydrophobic (C4) column (see Supporting Information). Conditions for RP-HPLC purification were as follows: a Zorbax C18 column (9.4 mm ×250 mm) at a flow rate of 4 mL/min or a Microsorb MV-C4 column (4.6 mm ×250 mm) at a flow rate of 1 mL/min, with a linear gradient from 5% to 50% acetonitrile and an aqueous buffer consisting of 0.1 M triethylammonium acetate, pH 7.0. Elution was monitored by diode array detection at 260 nm. Oligonucleotides were desalted using C18 Sep-pak cartridges (Waters) and analyzed by MALDI-TOF-MS. Thermal Melting Studies. Absorbance versus temperature profiles (melting and annealing curves) were recorded on a Cary 3 Bio UV-visible spectrophotometer with a 6 × 6 multicell block Peltier temperature controller (Varian, Inc.). Oligonucleotides were dissolved in buffer containing 100 mM NaCl, 10 mM Na2HPO4, and 0.1 mM Na2EDTA, pH 7. Samples were denatured by raising the temperature to 90 °C for 10 min prior to the beginning of an experiment. Melting and annealing experiments were conducted in two stages by starting the experiment at 90 °C, cooling to 5 °C, heating from 5 °C, and ending at 90 °C. Absorbance was measured at 260 nm with a heating/cooling rate of 0.5 °C/min and data collection at 0.2 °C intervals. Data Analysis. The Tm data obtained were exported to the software application Microcal Origin (OriginLab Corporation, Northampton, MA). Sigmoidal curves were obtained using Boltzmann logged data-fit function, and the melting temperature was obtained by fitting the first derivative of the sigmoidal curve to a Gaussian function to identify the center of the peak, which is the Tm, or melting temperature. Calculation of Thermodynamic Parameters. Thermodynamic parameters were determined from averages of fits from individual melting curves using the van’t Hoff calculation in the Cary WinUV Thermal software (Varian, Inc.). The parameters were derived by van’t Hoff analysis for self-complementary sequences where a two state model is assumed for the helix-coil transition using the method described by Puglisi and Tinoco (36). The Tm plot (absorbance vs temperature) was converted to an alpha (R) plot (fraction of molecules paired vs temperature) using the equation: R)

A - As Ad - As

(1)

where A is the absorbance at given temperature, and As and Ad are the absorbance values for the single- and double-stranded states, respectively. The parameter R refers to the fraction of

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molecules paired. The upper and lower baselines of the Tm plots were fitted using linear least-squares fits to obtain a good approximation for As and Ad. The thermodynamic parameters ∆H, ∆S, and ∆G were determined by generating a van’t Hoff plot (ln K vs 1/T) using values of K calculated at each temperature from the R plot using the following equation: K)

R 2(1 - R)2Ct

(2)

where Ct is the total strand concentration. The thermodynamic parameters can be derived directly from the van’t Hoff plot in which the slope is -∆H°/R, and the intercept is ∆S°/R. The free energy change (∆G°) at any temperature can be determined by using the following relationship: ∆G° ) ∆H° - T∆S°

(3)

Circular Dichroism Spectroscopy. CD spectra were recorded on a JASCO circular dichroism spectropolarimeter. Oligonucleotide samples were prepared in a buffer containing 100 mM NaCl, 10 mM Na2HPO4, and 0.1 mM Na2EDTA, pH 7. Samples were scanned over the wavelength range of 700 to 225 nm at a rate of 20 nm/min with time constant and bandwidth parameters set at 2 s and 1 nm, respectively. Data were collected at every 0.2 nm increment for each scan, and an average of 3 scans was used to generate each data set. A baseline scan (average of 3 scans) was collected with buffer only prior to each scan, and the baseline was subtracted from the sample scans. Fluorescence Microscopy. Mouse embryo fibroblasts immortalized with SV-40 large T antigen were seeded into a 24well plate containing one glass coverslip per well in 1.0 mL of Dulbecco’s modified Eagle’s media (DMEM) containing 10% fetal bovine serum and supplemented with 1% L-glutamine and 1% penicillin/streptomycin. Cells were incubated overnight in a humidified chamber at 37 °C in an atmosphere containing 5% carbon dioxide. Twenty-four hours after plating, media were aspirated from the wells, and fresh media (0.5 mL) containing the indicated reagents were added and cultures returned to the 37 °C incubator. After the indicated time points, cell monolayers were rinsed three times with 1 mL of phosphate buffered saline (PBS) per well, fixed in methanol, and mounted on glass slides. Fluorescent images were obtained using a Nikon Eclipse microscope.

RESULTS AND DISCUSSION Design and Synthesis. The design of the ON-lipid conjugates and the choice of the lipophilic alkyl chain lengths examined here were guided by considering (i) the average distance between the 3′-O and 5′-O of a B-DNA duplex (determined to be 16.2 Å (21)), (ii) the hydrophobicity of the appending group, and (iii) the ease of synthesis. Molecular models were constructed and demonstrate that an interaction is likely to occur between the terminal C12 alkyl groups and with the terminal base pairs in an energy-minimized conformation (Figure 3, water molecules not displayed for clarity). Modeling of the sequence 10A, with 5′-C12 and 3′-C12OH modifications (Figure 3A-D), indicated that there are potentially favorable hydrophobic interactions formed between the C12 alkyl chains, which span the distance between the terminal 3′-O and 5′-O and the planar surface of the terminal base pairs. These hydrophobic interactions are expected to shield the terminal hydrogen bonds from solvent water molecules while releasing water molecules in an entropically favorable manner resulting in duplex stabilization.

Figure 3. Energy minimized models: (A) 5′-C12 modified 10A; perspective view perpendicular to the helical axis showing the interaction of 5′-C12 with the terminal base pair. (B) 5′-C12 modified 10A: perspective view down the helical axis. (C) 5′-C12 and 3′- C12OH modified 10A: perspective view perpendicular to the helical axis showing the interaction of 5′-C12 and 3′-C12OH with the terminal base pair. (D) 5′-C12 and 3′-C12OH modified 10A: perspective view down the helical axis showing the interaction of 5′-C12 and 3′-C12OH with each other.

The anionic DNA conjugated to a hydrophobic molecule raises the possibility of micelle formation. It is intuitive and also shown experimentally (22) that there is a direct correlation between alkyl chain length and the propensity for forming micelles. On the basis of our models, we found the C12 alkyl group to have the optimal length (16.4 Å) for spanning the distance between the terminal base pair and that this length should exhibit a lower propensity for micelle formation in comparison to longer alkyl chains or more hydrophobic groups such as cholesterol. It has been shown that the critical micelle concentrations (CMC) for cholesterol-conjugated 10-mers were greater than 150 µM, and in some cases, no micelles were formed at concentrations less than 1 mM (10, 23). The C12 conjugates used in our studies are less hydrophobic than cholesterol and were studied at a concentration of 10 µM, which is significantly below the reported CMC for cholesterol conjugated 10-mers. Hence, no micelle or liposome formation was anticipated or observed. The syntheses of C12 modified 10-mer sequences were achieved by solid phase methodologies using standard automated protocols and reagents on an ABI 394 DNA/RNA synthesizer. C12 lipophilic monomers for 5′-end attachment were prepared by synthesis of the phosphoramidite derivative of 1-dodecanol (2) for the synthesis of DNA with 5′-C12 modification. The dimethoxytrityl (DMT) phosphoramidite derivative of 1, 12dodecanediol (4) was prepared for the synthesis of DNA with 5′-C12-OH modification after DMTr cleavage and deprotection (Scheme 1). The CPG support bearing the C12 monomer (9) was prepared as described (Experimental Procedures) for the 3′-C12-OH modified DNA (Scheme 1). Thermal Melting Studies. The effects of C12 terminal modifications on duplex DNA stability were assessed by thermal melting (Tm) studies. Self-complementary 10-mer sequences were synthesized bearing C12 on the 5′-and/or 3′-termini. The sequences had 60% GC content with one sequence (10A) having an A · T terminal base pair and a second sequence (10B) having a C · G terminal base pair in order to compare the effect of hydrophobic group stacking over C · G vs A · T bases. The results show a general trend of increasing Tm value with increasing sequence hydrophobicity as measured by retention on a C18 reversed phase HPLC column (see later sections). C12 or C12OH added to the 5′-end of sequence 10A (A · T terminal base pair) increased the Tm value by 4.8 °C over the unmodified

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Scheme 1. Preparation of C12 Phosphoramidites and Solid Support for the Synthesis of C12 Modified DNA

Figure 5. Thermal profile for self-complementary duplex 10B: (5′CAGTCGACTG-3′)2. The thermal profile shows increased Tm values with the addition of C12 substituents: ∆Tm (3′C12-OH) ) +0 °C, ∆Tm (5′C12-OH) ) +5.3 °C, ∆Tm (5′C12) ) +8.2 °C, and ∆Tm (5′ C12+3′ C12-OH) ) +12.3 °C.

sequences, whereas C12 added to both the 3′- and 5′-termini increased the Tm value by 9.8 °C. Addition of C12 to the 3′end did not exhibit any appreciable effect on Tm value (Figure 4). Similar results were obtained for sequence 10B (C · G terminal base pair) with an increase of 8.2 and 5.3 °C for 5′end modification with C12 and C12-OH, respectively. An increase in Tm of 12 °C was observed for C12 added to both 5′- and 3′-ends, and no appreciable difference was observed upon 3′-end modification only (Figure 5). The thermodynamic parameters (∆H, ∆S, and ∆G) were extracted from Tm plots as described in Experimental Procedures (Tables 1 and 2). Free energies were calculated at 25 and 37 °C and showed that stabilization of the duplex correlated with

Figure 4. Thermal profile for the self-complementary duplex 10A: (5′ACGTCGACGT-3′)2. The thermal profile shows increased Tm values with the addition of C12 substituents: ∆Tm (3′C12-OH) ) +0 °C, ∆Tm (5′C12) ) ∆Tm (5′C12-OH) ) +4.8 °C, and ∆Tm (5′+3′ C12) ) +9.8 °C.

the measured increase in Tm values. The relative stabilities observed also correlated with the order of sequence hydrophobicity as measured by C18 reverse phase HPLC in the order: unmodified < 3′-C12-OH modified < 5′-C12-OH modified e 5′-C12 modified < 5′- and 3′-C12 modified. An exception to the trend occurs with the 3′-C12-OH modified sequences where no increase in Tm or stabilization was observed with the increase in hydrophobicity. This deviation from the trend is apparently not related to the hydroxyl group present at the end of the alkyl chain since the 5′-C12-OH modification confers increased Tm and stabilization to the sequences. It has been shown previously that hydrophobic, aromatic molecules such as napthalene diimide (24, 25) and N,Ndimethylstilbene dicarboxamide (26, 27) stabilized DNA duplexes through π-π stacking interactions with the terminal DNA bases. Our molecular models predict the possibility of CH-π interactions between the hydrophobic alkyl groups and the terminal base pairs resulting in stabilization of the duplex and an increase in Tm. A study by Isaksson and Chattopadhyaya (28) revealed a correlation between the stacking geometry of dangling terminal bases and duplex stabilization due to shielding of the terminal hydrogen bonds from external water molecules by a single base overhang. In addition to geometry, the degree of stabilization is determined by the size of the overhanging base, the nature of the terminal base pair (A · T, G · C, T · A, or C · G), and the hydrophobicity of the overhanging base. As observed here stabilization afforded by hydrophobic groups is significantly greater than the resulting stability of adding a one base overhang at each end of the sequence or the addition of either a C · G or A · T to both ends of the duplex (Tables 1 and 2). This can be attributed to better shielding of the hydrogen bonds of the terminal base pairs from the solvent due to the hydrophobic nature of the C12 alkyl group. A possible explanation for the lack of stabilization by C12 appended to the 3′-end may be due to the helical and stacking characteristics of the B-DNA (28, 29). The 2′-endo-3′-exo puckering mode of B-DNA translates into helical and stacking characteristics that results in an overhanging moiety (nucleobase or other conjugates) stacking better when attached to the 5′-end than when the same moiety is attached to the 3′-end (28, 30). Finally, stacking of the C12 group over a C · G base pair provides a greater degree of stabilization than stacking over an A · T base pair. Circular Dichroism. Circular dichroism experiments were performed to determine the effects of our hydrophobic modifications on the structures of the resulting 10-mer DNA duplexes (Figure 6). The results shown in Figure 6 indicate that there is

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Table 1. Thermodynamic Data Derived from Tm Curves for Self Complementary Sequence 10A Having an A · T Terminal Base Pair sequence (5′-3′)

∆H (kcal/mol)

∆S (cal/mol/K)

∆G(25 °C) (kcal/mol)

∆G(37 °C) (kcal/mol)

Kd (nM)

Tma (°C)

Tmb (°C)

ACG TCG ACG T TAC GTC GAC GT TAC GTC GAC GTA [C12]-ACG TCG ACG T [C12-OH]-ACG TCG ACG T ACG TCG ACG T-[C12-OH] [C12]-ACG TCG ACG T-[C12-OH]

-65.2 ( 1.6 -50.4 ( 3.7 -24.3 ( 3.7 -59.7 ( 1.6 -69.1 ( 1.4 -68.6 ( 1.1 -60.2 ( 1.0

-177.4 ( 5.0 -131.2 ( 11.1 -168.3 ( 11.1 -158.2 ( 4.6 -185.7 ( 4.1 -187.5 ( 3.5 -157.1 ( 2.6

-12.4 ( 0.2 -11.3 ( 0.4 -12.7 ( 0.4 -12.5 ( 0.2 -13.7 ( 0.1 -12.6 ( 0.4 -13.3 ( 0.2

-10.2 ( 0.1 -9.8 ( 0.5 -10.7 ( 0.5 -10.7 ( 0.2 -11.5 ( 0.1 -10.4 ( 0.4 -11.5 ( 0.2

58.9 128 28.2 29.3 8.1 46.7 8.1

52.7 ( 0.5 53.4 ( 0.3 55.9 ( 0.3 56.8 ( 0.4 57.5 ( 0.4 52.7 ( 2.0 61.2 ( 0.5

52.3 ( 0.5 53.3 ( 0.2 54.8 ( 0.2 57.1 ( 0.3 57.2 ( 0.4 50.3 ( 1.9 62.1 ( 0.3

a Tm determined from the R plot. b Tm determined by the derivative method. Tm values were measured in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl and 0.1 mM EDTA. Data are the average and SD of at least 3 experiments.

Table 2. Thermodynamic Data Derived from Tm Curves for Self Complementary Sequence 10B Having an C · G Terminal Base Pair sequence (5′ -3′)

∆H (kcal/mol)

∆S (cal/mol/K)

∆G(25 °C) (kcal/mol)

∆G(37 °C) (kcal/mol)

Kd (nM)

Tma (°C)

CAG TCG ACT G TCA GTC GAC TG TCA GTC GAC TGA [C12]-CAG TCG ACT G [C12-OH]-CAG TCG ACT G CAG TCG ACT G-[C12-OH] [C12]-CAG TCG ACT G-[C12-OH]

-72.6 ( 2.2 -83.0 ( 2.0 -67.0 ( 1.1 -71.4 ( 0.9 -68.5 ( 1.3 -58.1 ( 0.9 -70.2 ( 1.1

-201.8 ( 7.9 -235.4 ( 6.1 -183.7 ( 3.2 -193.7 ( 3.1 -186.5 ( 3.9 -158 ( 2.0 -187.6 ( 2.3

-12.4 ( 0.7 -12.8 ( 0.2 -12.3 ( 0.1 -13.7 ( 0.4 -12.9 ( 0.1 -11 ( 0.4 -14.3 ( 0.5

-10.0 ( 0.7 -10.0 ( 0.2 -10.1 ( 0.1 -11.4 ( 0.5 -10.7 ( 0.1 -9.1 ( 0.4 -12 ( 0.5

93.8 93.8 80.4 9.5 30.5 365 3.2

48 ( 0.6 47.8 ( 0.1 51.4 ( 0.2 55.8 ( 1.0 54.0 ( 0.0 47.0 ( 0.3 59.7 ( 0.5

Tm

b

(°C)

48.3 ( 0.5 47.3 ( 0.4 50.5 ( 0.1 56.5 ( 0.8 53.6 ( 0.0 46.2 ( 0.5 60.3 ( 0.5

a Tm determined from the R plot. b Tm determined by derivative method. Tm values were measured in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl and 0.1 mM EDTA. Data are the average and SD of at least 3 experiments.

Figure 6. Circular dichroism spectra illustrating that the DNA duplex is unperturbed by the addition of C12 and retains the B-form helical structure.

no deviation from a canonical B-DNA helix, with diagnostic positive signals observed between 265 and 285 nm and a negative signal between 250 and 260 nm. Oligonucleotides bearing modifications that retain a B-DNA duplex structure have the ability to bind proteins and the potential to be used in cocrystallization studies with DNA binding proteins as well as dsDNA transcription factor decoy agents for use as biological tools or therapeutic agents. HPLC As a Measure of Hydrophobicity. The retention time of C12 modified DNA sequences on a C18 reverse phase HPLC column provides an approximate indication of the relative hydrophobicity of the sequences (Figure 7). The relative hydrophobicity increases in the following order: unmodified (rt: 4.48 min) < 3′-C12-OH modified (rt: 7.04 min) < 5′-C12-OH modified (not shown) < 5′-C12 modified (rt: 8.93 min) < 5′C12 and 3′-C12-OH modified (9.62 min). The 3′-C12-OH group is less hydrophobic than the 5′-C12-OH, possibly due to the added length and flexibility associated with the 5′-CH2. These HPLC derived hydrophobicity parameters were used to correlate the relative hydrophobicity of these sequences to the biophysical properties associated with the modifications.

Figure 7. C18 reversed phase HPLC assessment of the relative hydrophobicities of each modified sequence. Hydrophobicity increased with successive additions of C12 to the termini as indicated by the increased retention time on a C18 column.

Cell Uptake Studies. The cell uptake of C12-modified dsDNA of the type shown in Figure 1 (having C12 conjugated to both the 3′- and 5′-termini and the opposite termini covalently linked via a C12 end-cap - Supporting Information) was assessed by fluorescence microscopy (Figure 8). Sequences were labeled at the 5′-end with DyLight DY647 fluorescent dye with one sequence (NFκB-Dye) having the C12 end-cap but lacking the 3′- and 5′-C12 modifications as a control. Mouse embryo fibroblast cells were incubated with 0.5 µM and 1 µM fluorescently labeled oligonucleotides for 1, 2, and 4 h time points. The results show a significant increase in cell uptake compared to that of the control sequence at both 0.5 µM and 1 µM oligonucleotide concentrations after 1 h with increased accumulation in the cytoplasm and nucleus as a function of incubation time.

CONCLUSIONS Much attention has been given to ON-lipid conjugates, particularly cholesterol, for enhancing the cell uptake of antisense oligonucleotides. In the context of double-stranded oligonucleotides, studies on small interfering RNA (siRNA) conjugated to cholesterol, derivatives of lithocholic acid, and

Lipid Modified Double-Stranded Oligonucleotides

Bioconjugate Chem., Vol. 21, No. 8, 2010 1543

Fellowship to B.L. We thank Dr. David Thompson for helpful discussions on lipophilic groups suitable for DNA modifications. Supporting Information Available: 1H, 13C, and 31P NMR spectra, and MALDI-TOF-MS data table for DNA sequences. This material is available free of charge via the Internet at http:// pubs.acs.org.

LITERATURE CITED

Figure 8. Fluorescence microscopy of mouse embryo fibroblast cells incubated with NFκB decoy oligonucleotide sequences labeled with DyLight DY647. NFκB-Dye: end-capped 16mer dsDNA. NFκB-C12Dye: end-capped 16-mer dsDNA bearing 5′-C12and 3′-C12-OH of the form described in Figure 1.

derivatives of 12-hydroxy lauric acid at the 5′-end of the sense strands were shown to be taken up into liver cells without the use of transfection reagents (31-33). This uptake is reported to be mediated by interactions with lipoprotein particles, lipoprotein receptors, and transmembrane proteins that selectively direct the uptake of siRNA into cells of the liver, gut, kidney, and steroidogenic organs (33). Indeed, siRNA conjugated to palmitic acid, oleic acid, and cholesterol showed no uptake into human fibrosarcoma cells (HT-1080) without the use of transfection reagents (34). In contrast to the more lipophilic groups used in these studies of dsRNA, we see efficient cell uptake of dsDNA with the conjugation of two C12 groups positioned adjacent to each other, mimicking the adjacent post-translational lipidation of proteins in nature that directs proteins to lipid rafts. It can be postulated from these results that, in addition to the relative lipophilicity of the molecules, the positioning of the conjugates relative to each other play a role in enhancing the cell uptake of ON-lipid conjugates. Further study is necessary to confirm the mechanism of cell uptake, which may also be cell type specific. In this article, we used a simple model to assess the effects of lipophilic groups attached to the termini of double-stranded DNA on the biophysical properties of the duplex, namely, their thermal stabilities. We have found that simple lipophilic alkyl groups attached to the termini of self-complementary sequences increases thermal stability in a manner indicative of (1) the occurrence of CH-π stacking interactions with the terminal base pair, (2) hydrophobic interactions between lipophilic groups on complementary strands of the duplex, and (3) effective shielding of the terminal hydrogen bonds from external water molecules. In comparison to the previous methods employed in stabilizing dsDNA by covalent cross-linking using more complex linker molecules, our current strategy offers a significant advantage in terms of simplicity and ease of synthesis. Taken together, these results may provide the basis for the development of a class of stable dsDNA that can be applied as biological tools or as transcription factor decoy inhibitors with improved stability and cell uptake properties for therapeutic purposes.

ACKNOWLEDGMENT This work was supported by a grant from the Indiana Elks Foundation, Walther Cancer Institute, and a Purdue Doctoral

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