Bioconjugate Chem. 2006, 17, 245−247
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Microstructured Liposome Array Barnabe´ Chaize,† Michel Nguyen,‡ Tristan Ruysschaert,† Ve´ronique le Berre,§ Emmanuelle Tre´visiol,§ Anne-Marie Caminade,‡ Jean Pierre Majoral,‡ Genevie`ve Pratviel,‡ Bernard Meunier,‡ Mathias Winterhalter,†,| and Didier Fournier*,† Institut de Pharmacologie et de Biologie Structurale, 205, route de Narbonne 31077 Toulouse Cedex, France, Laboratoire de Chimie de Coordination, 205, route de Narbonne 31077 Toulouse Cedex 4, France, Biochips Platforms of Genopole Toulouse Midi-Pyre´ne´es, UMR-CNRS 5504, UMR-INRA 792, Toulouse, France, and International University Bremen, P.O. Box 750 561, 28725 Bremen, Germany. Received September 9, 2005; Revised Manuscript Received November 24, 2005
Conversion of a DNA chip to a nanocapsule array was performed by grafting on a liposome an oligonucleotide complementary to an oligonucleotide bound to the array. Each liposome may be loaded by a soluble molecule or may present a hydrophobic or amphiphilic molecule inserted in its wall. To detect liposomes on the chip, we used fluorescent dyes encapsulated in the liposome internal volume or fluorescent lipids. We observed that an oligonucleotide-grafted liposome containing a defined dye specifically accumulated on the area where its complementary oligonucleotide had been spotted on the array. The virtually unlimited amount of addresses allows the specific binding of large amounts of liposomes in one single batch.
INTRODUCTION DNA microarrays or DNA chips allow us to analyze the expression of hundreds of genes in a single experiment (1). The technique consists of immobilizing a specific single stranded DNA sequence on a solid support using an automated spotting device. The obtained microstructured array is incubated with single stranded cDNA fragments obtained from RNA extracted from cells that are investigated. Complementary DNA fragments present in the solution will hybridize with the DNA linked to the array. Crossed correlation analysis of the hybridized DNA allows the discovery and the understanding of gene expression and metabolic pathway activation in organisms (2). This technology stimulated the development of other chips such as protein chips as diagnostic tools or as biosensors utilizing immobilized enzymes, antibodies, or membrane proteins (3, 4). However, protein structures are fragile. Immobilization with a harsh chemical treatment often leads to an uncontrolled decrease in activity and limits the use of this technology. The aim of this paper consists of using the nowadays wellestablished DNA microarray technology to allow immobilization of nanocontainers on the chip. Since proteins may be encapsulated in these containers in their native states, they remain in an ideal environment and resist protein denaturation due to dilution and to digestion by proteases (5, 6). Hydrophobic or amphiphilic molecules such as membrane proteins may be inserted in the nanocapsule wall. Thus, conversion of a DNA chip to a nanocontainer chip may result in the conversion of a DNA chip to a protein chip (Figure 1).
EXPERIMENTAL PROCEDURES Liposomes were prepared by hydration of a lipid film followed by freeze-thaw cycles and extrusion. 100 µL of 100 mM eggPC lipids (Lipoid, Ludwigshafen, Germany) in CHCl3 and 10 µL of 84 µM lipids DSPE-PEG(2000) Maleimide lipids * Corresponding author. † Institut de Pharmacologie et de Biologie Structurale. ‡ Laboratoire de Chimie de Coordination. § Biochips Platforms of Genopole Toulouse Midi-Pyre´ne´es. | International University Bremen.
Figure 1. Specific liposome attachment on the array via oligonucleotide hybridization. The specificity of the binding is guaranteed by the sequence dependent hybridization between the liposome grafted one and the one bound on the array.
(Avanti Polar Lipids, Alabaster, AL) in CHCl3 were driedin a 15 mL glass tube under N2 and then dried in a vacuum for 3 h. To identify liposomes on the array, we used a fluorescent probe. A lipid grafted rhodamine was used as dye for labeling the liposome membrane. We used 3 µL of 0.8 mM Rhod-PE lipids (Avanti Polar Lipids, Alabaster, AL) in CHCl3 in the formulation of the lipid film. As a soluble probe, we encapsulated the water-soluble fluorescent dye Cy5. The encapsulation was performed by freeze-thaw cycles in a buffer solution containing the probe to encapsulate (6, 7). 100 µL of 4.4 mM Cy5 fluorescent dye (Molecular Probes, Eugene, OR) in 2xSSPE (20 mM sodium phosphate buffer pH 7.4, 300 mM NaCl, 2 mM EDTA) was added and firmly vortexed to peel off and
10.1021/bc050273p CCC: $33.50 © 2006 American Chemical Society Published on Web 12/27/2005
246 Bioconjugate Chem., Vol. 17, No. 1, 2006
Technical Notes
Figure 2. Structures of dendrimer and oligonucleotides used.
dissolve the dried lipid film. The liposome suspension was frozen and thawed 30 times successively dipping the glass tube in liquid N2 and a 37 °C water bath. Then the volume of the solution was increased to 10 mL with 2xSSPE, and the solution was extruded through a 200 µm membrane filter (Schleicher & Schuell, Dassel, Germany) to calibrate the liposomes. At last, nonencapsulated probe was removed by gel filtration using a desalting column previously equilibrated with 2xSSPE (PD10, Amersham Biosciences, Uppsala, Sweden). To perform anchoring of oligonucleotides on the liposome surface, maleimide functionalized lipids were added in the lipid composition of the liposome membrane (1/100). The maleimide chemical function exhibited outside the liposome reacted with thiol chemical functions present at the 3′ end of the 27-base oligonucleotides A′ and B′ (Figure 2). The anchoring reaction occurred after 1 h at room temperature in 2xSSPE (1/1, thiol/ maleimide group). The maleimide grafted lipids that had not reacted thus far with the oligonucleotides were inactivated with an excess of cystein. 10 µL of 2 mM N-acetylcystein (Sigma, St. Louis, MO) in H2O was added to the liposome suspension, and the resulting mixture was incubated under agitation for 1 h. As DNA chips, we used dendrislides, which showed better sensitivity than classical slides; they were prepared as described (10, 11). Briefly, microscope glass slides were cleaned with an alcoholic solution of NaOH (2.5 M in alcohol/water: 3/2, v/v), rinsed with water, and silanized with (3-aminopropyl)triethoxysilane. The silanized glass slides were activated by KOH, then rinsed by water, and incubated in a solution containing the generation 4 of a phosphorus dendrimer harboring aldehyde functions at its surface (Figure 2) (58 µM) in tetrahydrofuran. The slides were then washed with tetrahydrofuran and with ethanol. Then 27-base oligonucleotides modified with an amine function at their 3′ extremities were spotted onto this dendrimer surface and reacted with their aldehyde functions. Oligonucleotides A, B, and C (Figure 2) were spotted on the support as for standard DNA chips, A and B were complementary to oligonucleotides grafted to the lipid containers, and C was used as control. An automated spotting device (Chip Writer Pro from Bio-Rad) allowed attaching 200 fmol of oligonucleotides on the support in a 150 µm diameter spot. The imines function formed between probes and dendrimers, aminosilane and dendrimer, as well as the unreacted aldehyde were reduced by immersion of the slides into an aqueous solution containing NaBH4 at 3.5 mg/mL, then washing with water during 5 min (3 times), and then drying under a stream of N2 or by centrifugation (800 rpm, 8 min). To bind liposomes on the slides, we took advantage of the complementarity between oligonucleotides grafted on liposomes and oligonucleotides spotted on the dendrislide. Liposome solutions were incubated overnight on the spotted coated glass slides. 1 mL of the liposome suspension (10 µM eggPC) was added to 9 mL of 2xSSPE and overnight incubated at 37 °C with the slide in a Petri dish. Following two washings with the same buffer, the reading of the fluorescence on the array was
Figure 3. Fluorescence image of bound liposomes on a DNA chip. To demonstrate the selectivity of the process, two examples treated in a slightly different manner are shown. The first slide (parts 3a-c) was functionalized with three different oligonucleotide spots: the upper one contains a random sequence, the middle one the complement to the red labeled (Cy5 at 635 nm) liposomes, and the bottom one the complementary nucleotides for the green labeled (rhodamine labeled lipids at 532 nm) liposomes. Clearly outside the oligonucleotide spots, binding of labeled vesicles was negligible. (a) Visualization by excitation at 635 nm of the red liposomes alone revealed an intensive red spot in the middle but also two less intense but still clear spots. (b) The same slide but with exciting (at 532 nm) of only the green color. Here, in addition to the expected main spot at the bottom, a small background is visible for the random sequence. (c) Overlay of both colors. (d) Increasing washing time improved the selectivity. We spotted on the left side oligonucleotides for green liposomes and in the middle four corresponding to the red liposomes; in the four spots with random sequence no color is revealed.
performed with a laser scanner (GenePix 4000A from Axon Inc.) at 532 and 635 nm, at appropriate sensitivity levels of the photomultiplier.
RESULTS AND DISCUSSION Liposomes containing Rhod-PE lipids were grafted with oligonucleotide A, and liposomes grafted with oligonucleotide B were loaded with fluorescent dye Cy5. Both liposome solutions were mixed and laid down a glass slide spotted with three oligonucleotides, A complementary to A′, B complementary to B′, and C a noncomplementary oligonucleotide. Following hybridization and extensive washing, reading of the fluorescence on the chip revealed colored spots corresponding to the two fluorescent dyes used; hybridization created a welllocalized predefined liposome array (Figure 3). The red spots correspond to an accumulation of liposomes containing the soluble Cy5 dyes detected with an excitation at 635 nm. The green spots correspond to an accumulation of liposomes having the membrane cross-linked rhodamine dye, which were detected with an excitation at 532 nm. Each dye corresponds to a liposome preparation with a specific oligonucleotide grafted on the external surface of the lipid membrane. Each kind of liposome binds to the spots corresponding to the complementary oligonucleotide on the chip. Typically the signal detected over each spot was 4 times higher than the background for the red spots and 6 times higher than the background for the green spots. The digital combination of the images obtained by exciting at 635 and 532 nm gives a full vision of the localization of each kind of liposomes on the chip. This experiment confirms that oligonucleotide hybridization may be used to tether a liposome on a surface (8, 9). We show here that, using a DNA chip and the specificity of hybridization, it is possible to immobilize liposomes at specific positions. Each liposome may contain a specific molecule and potentially protein; this allows a general conversion of a DNA chip into another chip such as a protein chip. On the basis of our previous work, the encapsulation procedure can be generalized for a wide range of water-soluble proteins. Encapsulation provides a controlled local environment for the protein and may avoid the
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Technical Notes
spreading of enzymatic products into the solution by trapping in the container when the product is charged (5). In addition, this method should be useful for membrane bound proteins (receptors, channels) which need to be in a lipid environment to keep their conformation and activity. The use of liposomes appears to be an alternative to the microspotting of lipidic membranes on γ-aminopropylsilane coated slides (4). An advantage of using nanocontainers is the possibility to substitute liposomes by polymersomes, which are usually more stable. Furthermore, this process allows their specific binding in a single step because of the high specificity of the oligonucleotide hybridization. Oligonucleotide-grafted liposomes are very exciting building blocks to create networks and other superstructures. DNA provides a virtual addressing without limits. Earlier systems used competitive binding of oligonucleotide to DNA covered liposomes to probe for the presence of specific DNA fragments. However, the liposomes serve only as an amplifier due to their size (12). Others created a larger network, and cross-linking was observed via turbidity measurements. Here the presented system is different. The liposomes appear to be excellent carriers for molecules to design a new kind of chip. The use of oligonucleotides as a means of anchoring shows a very high specificity and simplifies the localization of molecules on the array. Then molecules can be directed to their assigned place on the array in a single incubation step. However, the possibility to detect chemical enzymatic reactions is still limited by the finding of a unique reaction substrate for all the spotted proteins, and a system to avoid the spreading of the detectable product on the surface of the array. Here again the use of containers could be an advantage to maintain proteins in a specific reaction environment and to trap the enzymatic reaction products.
ACKNOWLEDGMENT This work was supported by the CNRS program “Proteomique et ge´nie des prote´ines” and by the ACI nanosciencesnanotechnologies NN082.
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