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Lipophilic Oligonucleotides Spontaneously Insert into Lipid Membranes, Bind Complementary DNA Strands, and Sequester into Lipid-Disordered Domains Andreas Bunge,†,‡ Anke Kurz,‡,§ Anne-Kathrin Windeck,§ Thomas Korte,§ Wolfgang Flasche,| Ju¨rgen Liebscher,| Andreas Herrmann,§ and Daniel Huster*,† Junior Research Group “Structural Biology of Membrane Proteins”, Institute of Biotechnology, Martin Luther UniVersity Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany, Institute of Biology/ Biophysics, Humboldt-UniVersity Berlin, InValidenstrasse 42, D-10115 Berlin, Germany, and Institute of Chemistry, Humboldt-UniVersity Berlin, Brook-Taylor-Strasse 2, D-12489 Berlin, Germany ReceiVed NoVember 1, 2006. In Final Form: January 30, 2007 For the development of surface functionalized bilayers, we have synthesized lipophilic oligonucleotides to combine the molecular recognition mechanism of nucleic acids and the self-assembly characteristics of lipids in planar membranes. A lipophilic oligonucleotide consisting of 21 thymidine units and two lipophilic nucleotides with an R-tocopherol moiety as a lipophilic anchor was synthesized using solid-phase methods with a phosphoramadite strategy. The interaction of the water soluble lipophilic oligonucleotide with vesicular lipid membranes and its capability to bind complementary DNA strands was studied using complementary methods such as NMR, EPR, DSC, fluorescence spectroscopy, and fluorescence microscopy. This oligonucleotide inserted stably into preformed membranes from the aqueous phase. Thereby, no significant perturbation of the lipid bilayer and its stability was observed. However, the non-lipidated end of the oligonucleotide is exposed to the aqueous environment, is relatively mobile, and is free to interact with complementary DNA strands. Binding of the complementary single-stranded DNA molecules is fast and accomplished by the formation of Watson-Crick base pairs, which was confirmed by 1H NMR chemical shift analysis and fluorescence resonance energy transfer. The molecular structure of the membrane bound DNA double helix is very similar to the free double-stranded DNA. Further, the membrane bound DNA double strands also undergo regular melting. Finally, in raft-like membrane mixtures, the lipophilic oligonucleotide was shown to preferentially sequester into liquid-disordered membrane domains.
Introduction Membrane binding by covalently attached lipid modifications is a common biological motif for a significant portion of all cellular proteins.1 Covalent attachment of fatty acids provides a soluble molecule with sufficient hydrophobic character for permanent membrane insertion. In recent years, this biological mechanism of membrane binding has been successfully exploited for biotechnological applications. For instance, lipid-anchored RNA and DNA oligonucleotides have been developed for various purposes in nanobiotechnology, cell biology, and for therapeutic strategies in medicine.2-6 In particular, lipophilic oligonucelotides have been synthesized to improve cellular uptake of antisense single-stranded DNA and interference RNA (iRNA).2,3,7,8 By * Corresponding author. Tel.: +49 (0) 345-55-24942. Fax: +49 (0) 34555-27013. E-mail:
[email protected]. † Martin Luther University Halle-Wittenberg. ‡ A.B. and A.K. contributed equally. § Institute of Biology/Biophysics, Humboldt-University Berlin. | Institute of Chemistry, Humboldt-University Berlin. (1) Casey, P. J. Science 1995, 268, 221-225. (2) Boutorin, A. S.; Guœkova, L. V.; Ivanova, E. M.; Kobetz, N. D.; Zarytova, V. F.; Ryte, A. S.; Yurchenko, L. V.; Vlassov, V. V. FEBS Lett. 1989, 254, 129-132. (3) Shea, R. G.; Marsters, J. C.; Bischofberger, N. Nucleic Acids Res. 1990, 18, 3777-3783. (4) MacKellar, C.; Graham, D.; Will, D. W.; Burgess, S.; Brown, T. Nucleic Acids Res. 1992, 20, 3411-3417. (5) Bonaccio, S.; Walde, P.; Luisi, P. L. J. Phys. Chem. 1994, 98, 6661-6663. (6) Tomkins, J. M.; Barnes, K. J.; Blaker, A. J.; Watkins, W. J.; Abell, C. Tetrahedron Lett. 1997, 38, 691-694. (7) Krieg, A. M.; Tonkinson, J.; Matson, S.; Zhao, Q.; Saxon, M.; Zhang, L. M.; Bhanja, U.; Yakubov, L.; Stein, C. A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1048-1052. (8) Lorenz, C.; Hadwiger, P.; John, M.; Vornlocher, H. P.; Unverzagt, C. Bioorg. Med. Chem. Lett. 2004, 14, 4975-4977.
using a lipophilic RNA construct, efficient silencing of the endogenous apolipoprotein B in mice has been reported upon injection of short interfering RNAs (siRNAs) coupled to a cholesterol membrane anchor.9 In this way, lipophilic oligonucleotides could represent a new class of drug carriers for diagnostic and therapeutic applications. The main idea of these approaches is to take advantage of the base pairing that allows single-stranded oligonucleotides to bind with the highest possible specificity to a cellular target. To this end, lipophilic oligonucleotides with appropriate properties have to be developed. General prerequisites for an efficient application in various fields are an easy and flexible synthesis, efficient and stable membrane incorporation, and capacity to recognize and bind specific single-stranded nucleic acids. Several studies have demonstrated the potential of such lipophilic oligonucleotides to decorate and functionalize membranes (e.g., with specific ligands or vesicles attached to the complementary oligonucleotide strand). For example, vesicles containing lipophilic DNA strands could be tethered to planar bilayers displaying oligonucleotides of complementary sequence.10 By using an array of two different lipophilic oligonucelotides of orthogonal sequences on supported bilayers, sorting of vesicles was achieved according to the lipophilic complementary oligonucleotide incorporated in the vesicle membrane. Although this and other studies showed that lipophilic oligo(9) Soutschek, J.; Akinc, A.; Bramlage, B.; Charisse, K.; Constien, R.; Donoghue, M.; Elbashir, S.; Geick, A.; Hadwiger, P.; Harborth, J.; John, M.; Kesavan, V.; Lavine, G.; Pandey, R. K.; Racie, T.; Rajeev, K. G.; Rohl, I.; Toudjarska, I.; Wang, G.; Wuschko, S.; Bumcrot, D.; Koteliansky, V.; Limmer, S.; Manoharan, M.; Vornlocher, H. P. Nature 2004, 432, 173-178. (10) Yoshina-Ishii, C.; Boxer, S. G. J. Am. Chem. Soc. 2003, 125, 3696-3697.
10.1021/la063188u CCC: $37.00 © 2007 American Chemical Society Published on Web 03/17/2007
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nucelotides bind complementary strands, it is not known whether a double helix is formed, how the membranes affect the dynamics and structure of single- and double-stranded oligonucleotides, and if the membrane morphology and stability are compromised by oligonucleotide binding. Clearly, quantitative biophysical methods are called for to investigate the microscopic structure of such complexes. For various purposes, it would also be desirable to achieve distinct patterns of specific lateral organization of oligonucleotides in membranes. By selecting appropriate lipid anchors, enrichment of the lipophilic oligonucleotides in specific membrane domains should allow the creation of distinct functional compartments on a membrane surface. Such separations could be easily reversed just by increasing the temperature above a critical value. Local lateral recruitment of lipophilic oligonucleotides in membranes may also provide a strategy to improve cellular uptake, given the fact that endocytotic processes involve specific lipid domains (for a review, see ref 11). Essential problems related to the insertion of lipophilic oligonucelotides into existing membranes are associated with their hydrophobic (amphiphilic properties) and their supramolecular organization in aqueous solution (in the absence of membranes). Binding of oligonucleotides with one lipid anchor may be too weak as has been shown recently for cholesterol based anchoring of DNA.12 However, the stability of membrane binding has been shown to be improved by hybridization between two DNA oligonucleotides of different length, each being coupled to cholesterol.12 Other studies have also used oligonucleotides with two lipid anchors. Typically, alkyl chains have been linked to the oligonucleotide strands in close proximity. However, those constructs may tend to form stable supramolecular structures in aqueous solution, making it difficult to insert into preformed membranes.13 In biological systems, proteins are targeted to lipid membranes, for instance, by post-tranlational fatty acid modification. Two molecular patterns have evolved: (i) a double lipid modification (typically one palmitoyl and one farnesyl chain) or (ii) one lipid modification (typically a myristoyl chain) in combination with a cluster of basic residues on the protein that support the hydrophobic insertion of the chain by electrostatic interactions with the negatively charged inner leaflet of the plasma membrane.14,15 The first option appears to be the structural motif that could be exploited for the development of the biotechnological applications mentioned previously. For the present study, we recently synthesized a single-stranded DNA molecule that carries two hydrophobic anchors.16 The oligonucleotide (LT23mer) is a 23mer consisting of 21 thymidine (T) units and two lipophilic nucleotides L in positions 1 and 8 that were modified with R-tocopherol units as lipophilic anchors (Figure 1). Both anchors were separated by six nucleotides to reduce the stability of micellar organization and, by that, to improve insertion into membranes. For selected experiments, a molecule LT25mer was synthesized, which was akin to LT23mer but contained two more thymidine units at the 3′-end. We investigated the intercalation and organization of this lipidated (11) Parton, R. G.; Richards, A. A. Traffic 2003, 4, 724-738. (12) Pfeiffer, I.; Ho¨o¨k, F. J. Am. Chem. Soc. 2004, 126, 10224-10225. (13) Gosse, C.; Boutorine, A.; Aujard, I.; Chami, M.; Kononov, A.; Cogne´Laage, E.; Allemand, J.-F.; Li, J.; Jullien, L. J. Phys. Chem. B 2004, 108, 64856497. (14) Peitzsch, R. M.; McLaughlin, S. Biochemistry 1993, 32, 10436-10443. (15) Schroeder, H.; Leventis, R.; Rex, S.; Schelhaas, M.; Nagele, E.; Waldmann, H.; Silvius, J. R. Biochemistry 1997, 36, 13102-13109. (16) Kurz, A.; Bunge, A.; Windeck, A.-K.; Rost, M.; Flasche, W.; Arbuzova, A.; Strobach, D.; Mu¨ller, S.; Liebscher, J.; Huster, D.; Herrmann, A. Angew. Chem., Int. Ed. 2006, 45, 4440-4444.
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Figure 1. Chemical structure of the lipophilic building block L and the bases’ sequence of the molecule of LT23mer.
single-stranded DNA molecule in lipid membranes and its association with complementary olignucleotides. Using several techniques such as NMR, EPR, and fluorescence spectroscopy, differential scanning calorimetry (DSC), and fluorescence microscopy, we show that LT23mer stably inserts into lipid membranes, displays the oligonucleotide on the vesicular surface, binds complementary DNA strands by formation of WatsonCrick base pairs, and preferentially sequesters into liquiddisordered domains in membrane mixtures. Preliminary data were already published;16 the current paper represents novel experiments on the insertion, dublex formation, and distribution of LT23mer in lipid membranes and raft structures. Material and Methods Materials. 1-Palmitoyl-2-oleoyl-sn-glycerophosphocholine (POPC), 1-palmitoyl-d31-2-oleoyl-sn-glycerophosphocholine (POPC-d31), sphingomyeline (SM), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), and the fluorescent lipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-1,2-dipalmitoyl-snglycero-3-phosphatidyl-ethanolamine (N-NBD-PE) were obtained from Avanti Polar Lipids (Alabaster, AL). Hoechst 33342 (bisbenzimidazoles) was purchased from Serva (Heidelberg, Germany). Cholesterol and Merocynanine (M540) were purchased from Sigma (Deisenhof, Germany). DNA oligonucleotides without or with a covalently attached fluorophore (rhodamine, TAMRA) or spin label were synthesized by BioTez (Berlin, Germany). The spin-labeled oxyl-2,2,5,5,tetramethylpyrroline-3-carboxylate N-hydroxysuccinimide was obtained from Toronto Research Chemicals (Toronto, Canada) and was covalently linked to the 3′-terminus of a A20mer oligonucelotide. Synthesis of LT23mer. Synthesis of LT23mer was described recently.16 Briefly, the oligomer was synthesized on a DNA synthesizer applying the phosphoramidite strategy.17 The lipophilic tocopherylpropinylcytidine L (see Figure 1) was obtained by Sonogashira-coupling adopting a recently published procedure18 and transformed into the 5′-DMTr protected 3′-diisopropylaminocyanethylphosphoramidite by conventional methods. This method provides a high flexibility because lipophilic nucleotides can be introduced into any position of an oligonucleotide. Preparation of Liposomes. For preparation of large unilamellar vesicles (LUVs), the lipid mixture dissolved in chloroform was evaporated by a rotary evaporator forming a thin lipid layer on the flask wall. The lipid film was resuspended in aqueous buffer (80250 mM KCl, 10 mM Hepes, pH 8). Subsequently, LUVs were formed by subjecting this suspension to five freeze-thaw cycles and extrusion19 through a polycarbonate filter (Nucleopore GmbH, Tu¨bingen, Germany) of 100 nm diameter using an extruder (Lipex Biomembranes Inc., Vancouver, Canada). Giant unilamellar vesicles (GUVs) were generated at room temperature by an electroformation technique in a chamber made (17) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223-2311. (18) Flasche, W.; Cismas, C.; Herrmann, A.; Liebscher, J. Synthesis 2004, 2335-2341. (19) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55-65.
Lipophilic Oligonucleotide Actions of indium-tin-oxide coated glass slides.20,21 Lipids were dissolved in chloroform (0.25 mg/mL), and 70 µL of the lipid dispersion was deposited in small droplets on each slide. The solvent was evaporated in a desiccator (10 mbar) at room temperature for 60 min. Subsequently, the chamber was assembled by sealing both glass slides with silicon paste. An aqueous solution (250 mM sucrose, 10 mM HEPES, 0.02% NaN3) was injected with a syringe through a micropore filter immediately before connecting the completely sealed chamber to an electrical signal generator. LT23mer was added to the solution to allow its incorporation during the GUV preparation. The voltage of the applied AC field was increased in steps every 6 min from 20 mV up to 1.1 V while continuously increasing the frequency from 4 to 10 Hz within the first minute. The AC field was applied for 3-12 h. To complete the procedure, the voltage was raised to 1.3 V, and the frequency was lowered to 4 Hz. The chamber was then stored in a refrigerator at 4 °C. For washing and dilution of the vesicle suspension, equiosmolar buffers were used. Multilamellar vesicles (MLVs) were produced after POPC LUV samples containing LT23mer (100:1 molar ratio) in aqueous buffer (10 mM HEPES, 100 mM NaCl, pH 7.4) were ultracentrifuged (100 000g). Subsequently, the pellet was lyophilized and hydrated to 65 wt % D2O. The sample was homogenized by manual stirring and gentle centrifugation accompanied by several freeze-thaw cycles for equilibration. Finally, the sample was transferred into a 4 mm HR MAS rotor with Kel-F inserts (15 µL volume) for NMR measurements. NMR Spectroscopy. Static 31P NMR spectra of LT23mer containing MLVs were acquired on a Bruker DRX600 NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) at a resonance frequency of 242.9 MHz using a Hahn echo pulse sequence.22 A 31P 90° pulse length of 7 µs, a Hahn echo delay of 50 µs, a spectral width of 100 kHz, and a recycle delay of 3 s were used. Continuous-wave proton decoupling was applied during signal acquisition. Static 2H NMR spectra of MLVs in the absence and in the presence of LT23mer were recorded on a Bruker Avance 400 NMR spectrometer operating at a resonance frequency of 61.2 MHz for 2H using a solids probe equipped with a 5 mm solenoid coil. The 2H NMR spectra were accumulated at a spectrum width of 500 kHz using a phase-cycled quadrupolar echo sequence23 and a relaxation delay of 500 ms. The two 3 µs π/2 pulses were separated by a 60 µs delay. The 2H NMR spectra samples were dePaked24 and analyzed as described in detail in the literature.25 1H MAS NMR spectra were acquired on a Bruker DRX600 NMR spectrometer at a resonance frequency of 600.13 MHz using a 4 mm HR MAS probe at a MAS frequency of 6009 Hz. Typical π/2 pulse lengths were 8.9 µs. All 1H NMR spectra were referenced with respect to the terminal methyl group of the lipid chains of a POPC sample at 0.885 ppm. 2-D 1H MAS NOESY spectra26 of LT23mer containing MLV samples were acquired using a mixing time of 300 ms. The dwell time of the indirect dimension was set equal to one rotor period to avoid folding of spinning sidebands into the centerband region of the 2-D NOESY spectra. Typically, 400 complex data points were acquired in the indirect dimension with 32 scans per increment and a relaxation delay of 4 s. For high-resolution 1H NMR experiments in solution, aliquots of LT23mer were added to extruded 100 nm POPC vesicle suspensions (20 mM in D2O buffer (10 mM Hepes, 100 mM NaCl, pH 7.4)) at a LT23mer to POPC molar ratio of 1:100. NMR experiments were carried out on the Bruker DRX600 NMR spectrometer. Spectra were acquired at a spectral width of 7 kHz with a 90° pulse length of 10.3 µs. For phase sensitive NOESY experiments (mixing time (20) Angelova, M.; Soleau, S.; Meleard, P.; Faucon, J. F.; Bothorel, P. Prog. Lipid Res. 1992, 89, 127-131. (21) Mathivet, L.; Cribier, S.; Devaux, P. F. Biophys. J. 1996, 70, 1112-1121. (22) Hahn, E. L. Phys. ReV. 1950, 80, 580-594. (23) Davis, J. H.; Jeffrey, K. R.; Bloom, M.; Valic, M. I.; Higgs, T. P. Chem. Phys. Lett. 1976, 42, 390-394. (24) McCabe, M. A.; Wassall, S. R. J. Magn. Reson., Ser. B 1995, 106, 80-82. (25) Huster, D.; Arnold, K.; Gawrisch, K. Biochemistry 1998, 37, 1729917308. (26) Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. R. J. Chem. Phys. 1979, 71, 4546-4553.
Langmuir, Vol. 23, No. 8, 2007 4457 300 ms), 480 complex data points were collected in the t1 dimensions with 32 or 64 transients per increment at a 4 s relaxation delay. Subsequently, aliquots of A20mer were added at a 1:1 molar ratio with respect to LT23mer, and NMR spectra were collected again. All NMR experiments were carried out at 303 K. DSC. Differential scanning calorimetry measurements were carried out on a MicroCal VP DSC calorimeter (MicroCal, Inc., Northampton, MA) using a scan rate of 90 K/h. Only heating scans were performed. Aliquots of LT25mer were added to POPC LUVs (1 mg/mL), and the complementary A20mer strand was added to the solution after an incubation time of 30 min. The molar A20mer/LT25mer ratio was 1:1. The sample volume in the DSC cell was 0.5 mL. EPR Spectrsocopy. EPR spectra of spin-labeled oligonucleotides in the absence and in the presence of POPC LUVs (4 mM) were recorded at 25 °C using a Bruker ECS 106 spectrometer (Bruker, Karlsruhe, Germany) and the following parameters: modulation amplitude 2 G, power 20 mW, scan width 100 G, accumulation 6 times. The 3′-spin-labeled A20mer oligonucleotide was mixed with pure POPC liposomes or with POPC liposomes containing 1 mol % of LT23mer and incubated for 10 min at 25 °C. The molar ratio LT23mer/spin-labeled oligonucleotide was 100:1. The samples were filled into a 50 µL capillary tube (Corning, PYREX, Aldrich, Milwaukee, WI) and sealed for EPR measurements. The rotational correlation time τc (in s) was calculated according to27,28
(x )
τc ) 6.5 × 10-10∆H
Io -1 I-1
where ∆H is the width of the central EPR line (in G) and I0 and I-1 are the amplitudes of the central and high-field line, respectively. Fluorescence Spectroscopy. Fluorescence spectra and kinetics were recorded using an Aminco Bowman spectrometer series 2 (SLMAminco, Rochester, NY). N-NBD-PE was excited at 460 nm (slit width 4 nm), and fluorescence spectra were recorded between 470 and 610 nm with a scan rate of 1 nm/s, whereas for kinetic measurements, the emission wavelength was set to 532 nm (slit width 4 nm). A measure for the fluorescence resonance energy transfer (FRET) extent (ET) from N-NBD-PE (donor) to rhodamine-labeled oligonucleotides (acceptor) was quantified by ET ) 1 -
Fdonor-acceptor Fdonor
where Fdonor-acceptor and Fdonor are the fluorescence intensities of the donor in the presence and in the absence of the acceptor, respectively. The fluorescence spectrum of the DNA binding dye Hoechst 33342 was measured between 370 and 550 nm (slit width 4 nm) after excitation at 343 nm (slit width 4 nm). Fluorescence Microscopy. Fluorescence images of GUVs were obtained with an Olympus IX-81 inverted fluorescence microscope (Olympus, Hamburg, Germany) equipped with a cooled CCD camera (SPOT slider, Visitron Systems, Puchheim, Germany). Images were acquired using a 100× Plan-APO oil immersion objective with the appropriate differential interference contrast (DIC) optics and fluorescence filter sets: BP 330--385, FT 400, and LP 420 (Hoechst); BP 470--490, FT 505, and BP 510--550 (NBD, FITC); and BP 530--550, FT 580, and LP 590 (rhodamine, Merocyanine 540). Images were analyzed with the software Metavue and Metamorph (Universal Imaging, Downingtown, PA).
Results Incorporation of Lipophilic Oligonucleotides into Lipid Membranes. First, the interaction of the lipophilic oligonucleotide with lipid membranes was tested. Solid-state NMR techniques have proven to be useful to obtain information about the membrane (27) Morse, P. D.; Lusczakoski, D. M.; Simpson, D. A. Biochemistry 1979, 18, 5021-5029. (28) Keith, A.; Bulfield, G.; Snipes, W. Biophys. J. 1970, 10, 618-629.
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Figure 3. (A) Downfield region of a 600 MHz 1H MAS NOESY spectrum of LT23mer containing POPC LUVs at a mixing time of 300 ms and a temperature of 30 °C. Spectrum B shows the slice across the deoxyribose H1′ peak of thymidine taken from spectrum A. In spectrum C, a 1H MAS spectrum of POPC membranes is shown for comparison. Dashed lines indicate the most prominent cross-peaks of the thymidine moiety with the phospholipid headgroups, the chain methylenes, and the water. In trace D, the slice of a 1H NOESY spectrum of an aqueous solution of T20mer is shown for comparison.
Figure 2. (A) Proton decoupled 242.9 MHz 31P NMR spectrum of POPC multilamellar vesicles with LT23mer incorporated at a 100:1 molar ratio at 30 °C. (B) 2H NMR order parameter profile of POPC-d31 multilamellar vesicles in the absence (O) and in the presence (1) of 1 mol % LT23mer.
morphology and its modifications upon binding of molecules of varying size.29,30 A 31P NMR spectrum of POPC membranes in the presence of LT23mer (molar ratio 100:1) is shown in Figure 2A. The spectrum represents a superposition of an anisotropic line shape indicative of an axially symmetric chemical shift tensor characteristic for the liquid-crystalline lamellar phase state29 of the membrane and an isotropic line at 0 ppm of ∼1.3 ppm width. If one considers that the lipophilic oligonucleotide is very mobile at the membrane surface, the isotropic line in the 31P NMR spectrum could fully be attributed to the oligonucleotide phosphates. The relative integrals of both lines also roughly correspond to the correct mixing ratio of oligonucleotide to phospholipid. However, the isotropic contribution to the 31P NMR spectrum of LT23mer containing membranes could also indicate highly curved phospholipid structures induced by incorporation of LT23mer. We tested this possibility by accumulating 2H NMR spectra of POPC-d31 in the absence and in the presence of LT23mer. Here, the only contribution to the 2H NMR spectra comes from the lipid because LT23mer was not deuterated. These spectra only contained a small isotropic signal (less than 10% of the total lipid) in the presence of LT23mer, indicating that some POPC molecules would reside in highly curved phases. Nevertheless, these 10% lipids in non-lamellar phases could not make up for the large isotropic peak detected in panel A of (29) Seelig, J. Biochim. Biophys. Acta 1978, 515, 105-140. (30) Seelig, J.; Seelig, A. Q. ReV. Biophys. 1980, 13, 19-61.
Figure 2. It should also be noted that the preparation of the multilamellar vesicles necessary for 31P and 2H NMR measurements involved co-solubilization of lipid and LT23mer, which may have produced a small amount of non-lamellar phases. In all other experiments presented in this work, LT23mer was added directly to existing unilamellar liposomes through the aqueous phase. There was no indication for leakage or membrane rupture that would accompany the formation of non-lamellar-phase states. In addition, the 2H NMR spectra of POPC-d31 in the absence and in the presence of LT23mer yielded the lipid chain order parameters as a measure of the membrane distortion due to LT23mer incorporation. In spite of the bulky R-tocopherol units of LT23mer that represent the membrane anchor of each lipophilic oligonucleotide, only insignificant alterations of the membrane order were observed, indicated by almost identical order parameters (Figure 2B). This suggests that LT23mer incorporates well into lipid membranes and imposes only negligible alterations of the membrane structure. Next, we investigated the localization of the DNA bases of LT23mer with respect to the lipid membrane. For optimal base pairing with complementary DNA strands, the bases should be exposed to the aqueous phase. To study the localization of these molecular moieties, 1H MAS NOESY is a well-suited tool because intermolecular interactions that take place on short distance (
