Cholesterol-Linked Fluorescent Molecular

Bioconjugate Chem. 2006, 17, 1151−1155

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Cholesterol-Linked Fluorescent Molecular Beacons with Enhanced Cell Permeability Young Jun Seo,† Hyun Seok Jeong,† Eun-Kyoung Bang,† Gil Tae Hwang,† Jong Ha Jung,‡ Sung Key Jang,‡ and Byeang Hyean Kim*,† National Research Laboratory, Department of Chemistry, and Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31 Hyoja Dong, Pohang 790-784, Korea. Received March 29, 2006; Revised Manuscript Received July 29, 2006

We appended pyrene units covalently onto adenosine (forming AP units) and then incorporated them into oligonucleotides such that they were positioned in complementary locations in opposite strands in the middle positions of hairpin stems. System 1 (APAP) behaves as an effective molecular beacon (MB) that changes color from green to blue upon duplex formation. In addition, we attached a cholesterol unit to a free terminus of one of these hairpins; this approach enhanced the cellular delivery of the modified MB relative to those encountered when using conventional transfection methods. These structurally simple cholesterol-based MB systems, which can be synthesized very efficiently, have good potential for opening up new and exciting opportunities in the field of in vivo biosensors.

INTRODUCTION The detection of the molecular dynamics of DNA and RNA in living cells is becoming one of the most rapidly growing areas of research among all of the comprehensive investigations into the genomic information provided by nature (1). Such studies are aimed at providing information regarding the biological functions of DNA and RNA, the roles they play in disease, and their uses as (or interactions with) novel types of drugs. Fluorescence biosensors are powerful tools for probing the structures and interactions of biomolecules, but a strong demand exists for increasingly more advanced biomolecular recognition probes (2). We are interested in preparing chemically modified fluorescence biosensors, especially those exploiting π-π stacking interactions, that can also be used to investigate nucleic acid interactions and structures (3-5). One of the most useful aromatic planar dyes is pyrene, and its fluorescence has been utilized widely for detecting the DNA and RNA structures (6-8). Among the most useful probe systems for detecting molecular dynamics are the molecular beacons (MBs) (9-11). Unfortunately, these systems are somewhat limited for use as oligonucleotide probes in vivo, because they are difficult to transfect into cells due to their polar anionic backbones and bulky structures. Even though there is much demand for materials that enhance the transfection efficiencies of materials suitable for studying the dynamics of mRNA and DNA, the only means possible at present are the use of transfection agent, microinjection and electroporation techniques. General transfection techniques, such as those employing liposomes or dendrimers, often result in false positive signals and significantly increase the aggregation signal in the endosomes (12-14); microinjection (15) and electroporation (16) are invasive and may cause severe damage to cells. To overcome these difficulties, we have developed novel cholesterol-linked MBs. The cholesterol unit positioned at the 5′ * Corresponding author. National Research Laboratory, Department of Chemistry, Pohang University of Science and Technology, San 31 Hyoja Dong, Pohang 790-784, Korea. Fax: (82) 54-279-3399. Tel: (82) 54-279-2115. E-mail: [email protected]. † National Research Laboratory, Department of Chemistry. ‡ Department of Life Science, Division of Molecular and Life Sciences.

terminus of the hairpin MB allows these systems to enter into living cells efficiently without the need for any other transfection agent. Although there have been many research reports published in this area (17-22), to the best of our knowledge there are no papers describing cholesterol-containing MBs whose signal formation occurs through aromatic stacking within the stem of a hairpin. In this paper, we report such a system and demonstrate how it can be utilized effectively in vivo.

EXPERIMENTAL PROCEDURES Synthesis of Cholesterol Monomer Cholesterol-3-(2-cyanoethyl)-N,N-diisopropylphosphoramidite. Chloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine (225 mg, 0.95 mmol) was added to a solution of cholesterol (261 mg, 0.67 mmol) and 4-methylmorpholine (223 mg, 2.20 mmol) in CH2Cl2 (7 mL). After stirring the reaction mixture at room temperature for 5 min, the solution was washed with 5% aqueous NaHCO3. The organic layer was dried (Na2SO4) and concentrated under reduced pressure. Purification by flash chromatography (ethylacetate/hexane 1:7) provided the product as a white solid (277 mg, 70%). 1H NMR (300 MHz, CDCl3) δ ) 5.31 (m, 1H), 3.77 (m, 2H), 3.58 (m, 3H), 2.60 (t, J ) 6.3 Hz, 2H), 2.34 (m, 2H), 2.00-1.79 (m, 6H), 1.65-1.05 (m, 32H), 0.98 (s, 6H), 0.89 (d, J ) 6.3 Hz, 6H), 0.84 (d, J ) 6.5 Hz, 6H), 0.65 (s, 3H); 13C NMR (75.5 MHz, CDCl3) δ ) 140.8, 140.6, 121.8, 121.7, 117.6, 74.1, 74.1, 73.9, 73.8, 58.4, 58.2, 56.8, 56.2, 50.1, 43.1, 42.9, 42.3, 39.8, 39.6, 36.2, 35.8, 31.9, 28.3, 28.0, 24.8, 24.7, 24.5, 24.4, 24.3, 23.9, 22.9, 22.6, 21.1, 19.4, 18.8, 11.9; 31P NMR (121 MHz, CDCl3) δ ) 148.3, 148.2; IR (neat) υ ) 3749.8, 3734.4, 2964.7, 2962.3, 2902.5, 2867.4, 2252.8, 1465.0, 1457.6, 1363.7, 798.8, 731.1, 1183.0, 1060.1, 1031.4, 975.4, 798.8, 731.1 cm-1; HRMS (FAB) calcd for C36H63N2O2P 586.4627 [M + Na]+; found 609.4525. Synthesis of Oligonucleotides. The phosphoramidite AP and cholesterol were introduced as building blocks to produce fluorescent oligodeoxynucleotides (ODNs) on a controlled-pore glass (CPG) solid support by using the phosphoramidite approach and an automated DNA synthesizer (Perceptive Biosystems 8909 Expedite Nucleic Acid Synthesis System). For comparison, the unmodified ODNs were also prepared. The synthesized oligonucleotides were cleaved from the solid support upon treatment with 30% aqueous NH4OH (1.0 mL) for 10 h

10.1021/bc060078q CCC: $33.50 © 2006 American Chemical Society Published on Web 08/22/2006

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at 55 °C. The crude products from the automated ODN synthesis were lyophilized and diluted with distilled water (1 mL). The ODNs were purified by HPLC (Merck LichoCART C18 column; 10 × 250 mm; 10 µm; pore size: 100 Å). The HPLC mobile phase was held isocratically for 10 min with 5% acetonitrile/0.1 M triethylammonium acetate (TEAA) (pH 7.0) at a flow rate of 2.5 mL/min. The gradient was then increased linearly over 10 min from 5% to 50% acetonitrile/0.1 M TEAA at the same flow rate. The fractions containing the purified ODN were cooled and lyophilized. 80% aqueous AcOH was added to the ODN. After 30 min at ambient temperature, the AcOH was evaporated under reduced pressure. The residue was diluted with water (1 mL), and this solution was then purified by HPLC using the same conditions as those described above. The ODNs were analyzed by HPLC (Hewlett-Packard, ODS Hypersil; 4.6 × 200 mm; 5 m; 79916OD-574) using almost the same eluent system. For characterization, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric data of the fluorescent ODNs were collected using a PE Biosystems Voyager System 4095 instrument operated in the positive-ion mode and a 1:1 mixture of 3-hydroxypicolinic acid (0.35 M) and ammonium citrate (0.1 M) as the matrix; the accelerating voltage was 25 kV. Steady-State Fluorescence Measurements. Steady-state fluorescence (FL) spectra were recorded using an MD-5020 PTI model microscope photometer operating at a bandwidth of 15 nm and quartz cuvettes (0.5 × 2 cm) having a light pass length of 1 cm. The cell holder was thermostatted with circulating water that was controlled using a PolyScience digital temperature controller 9110. An excitation wavelength of 386 nm was used; all excitation and emission slits were set to 1 nm. All hybridizations and fluorescence experiments were performed in 1 mM MgCl2/100 mM Tris-HCl buffer (pH 8) containing a one-to-one ratio of complementary ODNs at a duplex concentration of 1.5 µM. Cell Culture and Transfection. Monolayers of human hepatoma cell line Huh7 were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco-BRL), 10% FBS (Hyclone), penicillin, and streptomycin. For fluorescence microscopy imaging, the coverslips were washed with HCl and rinsed with distilled water. 0.5% gelatin-coated coverslips were placed in 6-well dishes; culture cells were grown directly on the coverslips, with up to 80% confluence. Each ODN was diluted in serum-free DMEM to a final concentration of 40 nM. The cells were washed with phosphate-buffered saline (PBS); serum-free media containing each ODN (2 mL) were added to the cells grown on the coverslips. The lipofectamine 2000 reagent (Invitrogen) was used to compare the transfection efficiencies of the different delivery methods. Transfection assays were performed according to the manufacturer’s instructions. Image Acquisition and Processing. After incubation with an MB for the indicated time, the cells were washed three times with PBS and then fixed with 3.5% (w/v) paraformaldehyde (Sigma) at room temperature (RT) for 12 min. After being washed three times with PBS, the coverslips were placed on a glass slide and then sealed with transparent nail polish. The fluorescence images were captured using a cooled chargecoupled device camera and a Zeiss Axioplan microscope (Jena, Germany). Data were processed using Adobe Photoshop software (Mountain View, CA). Cellular Uptake of the Cholesterol-Bearing MB. The amount of ODN present within the transfected cells was quantified using a SPECTRAmax GEMINI microplate spectrofluorometer (Molecular Device). Huh7 cells were seeded onto 48-well culture dishes before transfection. 40 nM ODN solutions in serum-free DMEM were used in the transfection analyses. Cell extracts were collected after lysis of the cells (the cell lysis had been performed through the addition of lysis buffer). The fluorescence intensity was recorded with the excitation wave-

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Figure 1. Molecular beacons and their target DNA sequences. The underlined nucleotides are present in the loop regions for hybridizing with their complementary sequences.

Figure 2. Fluorescence spectra of 1 in their different states. (a) Emission of hairpin state 1; (b) emission of the duplex state 1‚2; All spectra were recorded using 1.5 µM solutions in buffer (100 mM trizma HCl/1 mM MgCl2; pH 8) at 25 °C; 386 nm.

Figure 3. Fluorescence spectra of (a) ODN 3 (λmax ) 509 nm) and (b) the perfectly matched duplex 3‚5 (445 nm). The conditions were the same as those described in Figure 2.

length set at 386 nm and the emission wavelengths set at 450 and 510 nm. Concurrently, the total cell mass was determined using the BCA method (Pierce, Rockford, IL).

RESULTS AND DISCUSSION Design of Functional Oligonucleotides. We synthesized pyrene-modified nucleoside building blocks through Sonogashira coupling of pyrene units to the 8-position of a 2′-deoxyadenosine base (i.e., to form 8-(1-ethynylpyrenyl)-2′-deoxyadenosine; AP) (23). The quantum yields of AP are very high: 81% and 89% in CHCl3 and MeOH (23), respectively. We incorporated AP into oligodeoxynucleotides (ODNs) by using the phosphoramidite method and the aid of a DNA synthesizer. We constructed modified single-stranded ODN 1 in which the two fluorophores were positioned in opposite strands of the stem of each hairpin. We also synthesized a perfectly matched sequence 2 for hybridizing to the loop regions of ODN 1. We synthesized a cholesterol-bearing MB system (ODN 3) that is complementary to ODN 5. We also synthesized the corresponding unmodified ODN 4 to compare the transfection properties between two systems (Figure 1). Cholesterol-Bearing MB. In Vitro Fluorescence Changes. Figure 2 displays the fluorescence spectra of APAP. Interestingly, 1 (existing in its hairpin state) emits a strongly red-shifted band

Fluorescent Molecular Beacons

Figure 4. Time-dependent cellular uptake of ODNs 3 and 4 in the absence and presence of lipofectamine (“lipo”). The fluorescence intensities were recorded with the excitation wavelength set at 386 nm and the emission wavelengths set at 510 nm. Concurrently, the total cell mass was determined using the BCA method (Pierce, Rockford, IL).

(λmax ) 521 nm; Figure 2a); in comparison, the value of λmax of the duplex 1‚2 is 450 nm (Figure 2b). On the basis of the results presented above, we prepared a cholesterol-bearing ODN incorporating a cholesterol unit at the 5′ end of the hairpin; because the 5′ and 3′ termini of ODN 1 are unsubstituted, it is very easy to use an ODN synthesizer to modify them with functional molecules. Thus, we synthesized a cholesterol-bearing MB system (ODN 3) that is complementary to the ODN 5. We also synthesized the corresponding

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unmodified ODN 4 to compare the two systems’ transfection properties. Figure 3 displays the change in the appearance of the fluorescence spectrum of ODN 3 after the addition of its target sequence, ODN 5. The spectra suggest that, even though we had attached a cholesterol moiety at the 5′ end of the MB, the photophysical signals of the MB system remained unaffected. We believe that the strongly red-shifted band at 509 nm must arise from stacking interactions between the two pyrene units of AP. In our fluorescence experiments. specific origin of this red-shifted band comes from an exiplex or merely that of a dimer of the two pyrene units. Fluorescence wavelengths are very close to that of a typical pyrene excimer (24), suggesting that the excited complex of each fluorophore was formed in the hairpin state. In ViVo Fluorometry. Our cholesterol-bearing MBs can enter into living cells efficiently without the need for any transfection agent (Figure 4). The transfection efficiency of the MB conjugated with cholesterol (ODN 3, blue curve) during the first 12 h in Huh7 cells was much higher than that of the unmodified MB (ODN 4, green curve); we observed essentially no signal for the latter, even after 12 h. For the sake of comparison, the unmodified ODN 4 was transfected with a commercial transfection reagent, lipofectamine (“ODN 4 + lipo,” red curve). The fluorescence obtained when using the transfection reagent (lipofectamine) was similar to that of the cholesterol-linked MB. In ViVo Fluorescence Microscopy. We performed a fluorescence microscopy experiment to observe the transfection pattern of the cholesterol-bearing MB in Huh7 cells. Figure 5 displays

Figure 5. Cellular delivery of MBs in Huh7 cells. (A-C) Fluorescence signals of cholesterol-conjugated ODN 3 transfected into Huh7 cells at different times (p.t.). Fluorescence signals of ODN 3 were not observed at 1 h p.t., but were detected in the cytoplasm around the nucleus at 6 and 12 h p.t. (D-F) Fluorescence of the unmodified ODN 4 after the indicated times (p.t.). The upper panels present the fluorescence signals of the MBs; the lower panels represent a combination of the images in the upper panels and the phase-contrast images of the cells.

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Figure 6. Cellular delivery of ODN 4 when using the lipofectamine reagent. (A-C) Fluorescence signals in Huh7 cells after transfection of the unmodified MB with lipofectamine for 12 h. Each emission filter accepted a different range of wavelengths: (A) 410-490 nm; (B) 508-523 nm; (C) >570 nm. (D) The three separate images from A-C (coded blue, green, and red, respectively) merged into a single image.

the cellular transfection property as imaged directly in vivo. Consistent with our fluorometry experiments, these images clearly demonstrate that the cellular transfection of the ODN 3 (bearing the cholesterol unit) is far better than that of ODN 4 (without cholesterol) and that obtained by using the conventional transfection agent (Figure 6). Indeed, in the case of ODN 4, we observed no signal, as expected, whereas ODN 3 displayed extremely strong signals in the cytoplasm. To demonstrate whether the ODN 3 reached the cytoplasm of cells, doublefluorescence staining experiments were carried out with Lysotracker (see Supporting Information). In the presence of the transfection reagent (lipofectamin), ODN 4 displayed different characteristics than did the cholesterol-linked ODN 3. Specially, the fluorescence signals obtained by using lipofectamin were concentrated in random “bright spots” in both the cytoplasm and the nucleus, indicating the aggregation pattern (Figure 6). We observed a similar trend after we transfected other oligonucleotides with lipofectamin in Huh7 cells (data not shown). However, we did not observe any cross-signal or nonspecific aggregated bright spots (compare Figure 5 to Figure 6) in our MB system. Therefore, our MB system is more efficient than one using the transfection agent. We are now investigating the ability of using such MB systems to fluorescently detect the dynamic pathway of hepatitis C virus in living cells.

CONCLUSION Systems 1 (APAP) individually exhibit aromatic stacking between the opposing pyrene units in the stems of their hairpins; their spectra display the characteristics of π-stacked pyrene units. Systems 1 (APAP) can be used as effective MBs that change color from green to blue upon duplex formation. These novel types of MBs are relatively simple to synthesize, and their termini remain free for the introduction of other useful functionalities. We introduced a cholesterol unit into one terminus of an MB and demonstrated clearly that the cellular delivery of this modified MB was enhanced significantly relative to those obtained by using the corresponding unmodified MB or by using conventional transfection agent-based methods. Cholesterol-

based transfection has the potential to avoid the endocentric pathway and, therefore, to reduce the number of false positive signals arising from nuclease degradation. The cholesterolbearing, pyrene-π-stacking-based MB system represents a new type of MB whose high cellular uptake efficiency makes such probes unique and novel; we believe that this one holds great promise for studying the dynamics of biomolecular systems in living cells and that its use will open up other new and exciting opportunities for applications in vivo.

ACKNOWLEDGMENT We are grateful to KOSEF for financial support through the National Research Laboratory Program (Laboratory for Modified Nucleic Acid Systems), Gene Therapy R&D program (M10534000011-05N3400-01110) and KNRRC program. BHK and SKJ are grateful to the National Core Research Center for Systems Bio-Dynamics. Supporting Information Available: MALDI-TOF mass spectra and confocal microscopy of synthesized compounds. This material is available free of charge via the Internet at http:// pubs.acs.org.

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