14544
J. Phys. Chem. B 2005, 109, 14544-14550
RNA Selectively Interacts with Vesicles Depending on Their Size Chris F. Thomas and Pier Luigi Luisi* Dipartimento di Biologia, UniVersita` degli studi Roma Tre, Viale Guglielmo Marconi 446, 00146 Rome, Italy ReceiVed: March 9, 2005; In Final Form: May 18, 2005
RNA and vesicles are two important molecular classes in the origin of life and early evolution, but they are not generally considered as interacting partners. The present paper reports about the interaction between tRNA (Esherichia coli) and vesicles made of the zwitterionic surfactant POPC (1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine), partially positively charged with small molar fractions (max 10%) of the singlechained CTAB (cetyltrimethylammonium bromide). CTAB is capable to insert efficiently in POPC vesicles (as determined by zeta-potential measurements), and the binding of tRNA to such charged vesicles operates a strong selection being critically dependent upon the vesicle size. The binding of tRNA to the vesicles is size-selective as it induces a strongly pronounced process of aggregation of large vesicles (ca. 160-nm diameter) but not of small ones (ca. 80-nm diameter) that are stable against vesicle aggregation (as followed by dynamic light-scattering and optical density measurements). The aggregation of the large vesicles is fully reversible upon the addition of RNase A. The selective behavior of tRNA with respect to differently sized vesicles is observable in separated samples as well as in a mixture of both populations. In the latter case, the fraction of large vesicles readily aggregates in the presence of the small ones that remain unaltered in the mixture. This kind of discrimination capability of RNA might have been of importance in the early phases of the formation of the protocells.
Introduction
Results and Discussion
Some particular aspects of the interaction between vesicles (spherical bilayer structures composed of amphiphilic molecules) and RNA have been considered by Yarus’ group. By applying selection and amplification cycles, these authors were able to detect lipid-binding RNA molecules on the surface of phospholipid vesicles.1-4 One of the aims of their approach was to investigate the effect of bound RNA on the vesicle membrane permeability and conductance, and the authors conclude that supramolecular RNA complexes indeed were capable of modifying bilayer properties. Other studies considered nuclear RNA5 and the effect of osmotic pressure on the membrane.6 However, the question whether RNA may display some specific sizedetermined interaction with vesicles remains unexplored in the literature. With the present work, we wish to specifically address such a question and to this aim we consider the interaction between tRNA and phospholipid vesicles made of POPC. We study the interaction between the vesicles and tRNA by application of dynamic light-scattering (DLS) technique so as to obtain information about the size and size distribution of the vesicles. Additionally, we follow the optical density (OD) of the samples. Since pure POPC vesicles did not undergo any observable changes upon the addition of tRNA solution, we introduced charges into the bilayer. These charged vesicles have been doped with small amounts of the positively charged cosurfactant CTAB so as to permit a Coulombic interaction with the negatively charged tRNA (taken from Esherichia coli). We detect the insertion of CTAB into the POPC bilayer by measuring the zeta potential, and the binding of tRNA to the positively charged POPC/CTAB vesicles is investigated by zeta potential measurements and fluorescence technique.
We will consider two distinct preparations of equally concentrated and composed vesicles (0.5 mM POPC/3.5% CTAB) with a narrow size distribution, as obtained by classic extrusion technique. (See Material and Methods, POPC/CTAB Vesicles, Methods i and ii. Method i describes extrusion of preliminarily mixed POPC/CTAB vesicles, whereas method ii stands for extrusion of pure POPC vesicles followed by subsequent addition of CTAB.) In particular, we will be dealing with a population of large vesicles extruded through polycarbonate filters of pore diameter 200 nm, which gives rise to a vesicle hydrodynamic mean diameter of around 160 nm as measured by DLS and a second population of small vesicles, centered at around 80-nm diameter, obtained with polycarbonate filters of 50-nm pore diameter. The mean diameter of a population of extruded vesicles can differ from the filter pore size.7 The experimental setup in our experiments was such that an excess of tRNA from a tRNA stock solution was added to a suspension containing the vesicles (final tRNA concentration 1.2 µM), and the resulting aggregation process was studied by DLS and OD measurements. Here, excess tRNA means that the tRNA concentration in terms of nucleotides/charges was higher than the concentration of CTAB that was introduced into the POPC matrix (see Materials and Methods). This roughly means a ratio of ca. 120 tRNA molecules per small vesicle and ca. 530 tRNA molecules per large vesicle, assuming monodisperse size distributions.8 I. Insertion of CTAB into POPC Vesicles. In a first experiment, the capability of CTAB to insert into POPC vesicles was detected by measuring the zeta potential of vesicles containing various molar fractions of CTAB. The aim was to follow the insertion of CTAB into the phospholipid bilayer and to investigate the electrophoretic behavior of the mixed vesicles.
* Author to whom correspondence should be addressed. Tel: 0039/06/ 55176329; fax: 0039/06/55176321; e-mail:
[email protected].
10.1021/jp0512210 CCC: $30.25 © 2005 American Chemical Society Published on Web 07/07/2005
RNA Selectively Interacts with Vesicles
Figure 1. Normalized zeta potentials for small (∼80 nm diameter) and large (∼160 nm diameter) 0.5 mM POPC vesicles as a function of CTAB molar fraction. CTAB was added externally from 10 mM CTAB solution to extruded POPC vesicles in 20 mM sodium phosphate buffer (pH 7.0), applied voltage 150 mV, sensed voltage ca. 130 mV.
The zeta potential (also known as the electrokinetic potential) for a charged particle suspended in a fluid arises from its electrokinetic motion when an electric field is applied. Because of the particle motion, the layer of ions and solvent molecules that surrounds the particle surface becomes distorted, which leads to a measurable net charge that is different from zero.9 The zeta potential is a complex variable and does not directly represent but corresponds to the charge on the particle surface. For this reason, a change in zeta potential upon addition of CTAB to POPC vesicles indicates the insertion of the cosurfactant into the phospholipid membrane. In Figure 1, normalized zeta potentials for small (diameter 80 nm) and large (diameter 160 nm) POPC vesicles as a function of the CTAB molar fraction are shown. The experimental setup was such that CTAB was externally added to the extruded POPC vesicles (see Materials and Methods, POPC/CTAB Vesicles, Method iii). For both the small and large vesicles, zeta potential increases upon the addition of CTAB indicating its uptake. Moreover, in the region of low molar fractions of CTAB, the zeta potential increase is in good approximation linear, in agreement with the results observed for other cosurfactants.10 When zeta potentials are reported in terms of normalized values, the curves are very close to each other. The absolute zeta potentials of the small vesicles turned out to be slightly lower than those of the large ones (less than 15% difference in average, data not shown). A similar observation has been made by Roy et al. with differently sized phosphatidylcholine/phosphatidylserine vesicles.11 Notice that the buffer concentration is relatively low and the dimension of the bilayer thickness, particularly for the small vesicles, becomes significant with respect to the vesicle diameter; this means that zeta potential is not independent of size anymore.11,12 As Menger et al. reported about NMR experiments that confirm quantitative insertion of similarly shaped anionic cosurfactants SDS (sodium dodecyl sulfate) and cationic CPB (cetylpyridinium bromide) into vesicles for small cosurfactant molar fractions,10 we consequently assume that little differences in absolute zeta potential values are due to the size dependence of the zeta potential. The molar concentrations of POPC and CTAB for both vesicle preparations are the same, leading to populations of approximately the same total surface area. Little effects brought about by the different curvature of the small and the large vesicles, however, are not supposed to significantly alter the surface charge density. In conclusion, the uptake of CTAB takes place in both cases with comparable efficiency. II. Aggregation of Large POPC/CTAB Vesicles. In the following section, we report about the aggregation of large vesicles from POPC/CTAB upon the addition of tRNA solution. As tRNA, we used the nonspecific mixture from E. coli as well as the specific tRNAPhe and obtained identical results. The
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Figure 2. Contrasting behavior of small (∼80 nm diameter) and large (∼160 nm diameter) 0.5 mM POPC/3.5% CTAB vesicles upon addition of tRNA (final concentration 1.2 µM) injected from a tRNA stock solution in 20 mM sodium phosphate buffer (pH 7.0). No aggregation of small vesicles, strong aggregation of large vesicles. Aggregation of the large vesicles is made reversible by addition of RNase A (final concentration 5 nM).
Figure 3. Size distribution of large vesicles from 0.5 mM POPC/3.5% CTAB in 20 mM phosphate pH 7.0 before addition of tRNA is shown as full line (-9-). After addition of tRNA, aggregates were formed (dashed line, -2-). After disaggregation by RNase A (concentration 5 nM), the size distribution (dotted line, -b-) very closely resembles the initial one. The difference in mean diameter (at the beginning 161.9 ( 2.2 nm, at the end 162.2 ( 3.4 nm) is within the standard deviation of the measurement.
vesicles for these experiments as well as for experiments in all following sections were produced by applying methods i and ii (see Materials and Methods, POPC/CTAB Vesicles). Figure 2 shows the optical density pattern recorded at λ ) 400 nm and gives evidence that addition of tRNA brought about a significant aggregation of the vesicles. In particular, DLS measurements of the aggregated sample show that the final size was more than ca. 1 µm (Figure 3) corresponding to assemblies formed by some hundreds of vesicles. The final aggregation state was reached 10 min after the addition of tRNA and was stable, as precipitation did not occur within at least 1 day. The aggregation process was completely reversed when RNase A (from bovine pancreas) was added to the aggregates (Figure 2). The optical density signal decreases rapidly to a value as low as the initial one. The transition from vesicles to aggregates and back can be followed also by DLS (Figure 3). It can be seen that upon addition of tRNA, the vesicles shift to a size distribution of large average size indicating the formation of aggregates. After the addition of RNase A, the vesicle size distribution shifts back to the position of the original one, centered at around ca. 160 nm. This suggests that the process of aggregation occurred without perturbing the vesicle properties. In a control experiment, a beforehand digested mixture of tRNA added to the vesicles did not give rise to any aggregation process at all, suggesting that only the intact tRNA molecule were capable to aggregate the vesicles. To exclude the occurrence of vesicle coalescence and leakage of the internal aqueous pools of the vesicles during the aggregation process, we entrapped the fluorophor calcein (10
14546 J. Phys. Chem. B, Vol. 109, No. 30, 2005
Thomas and Luisi
Figure 4. Linear relation between tRNA bound to the aggregates of 0.5 mM large vesicles (∼160 nm diameter) and the molar fraction of CTAB in POPC, indicating charge neutralization by electrostatic interaction.
TABLE 1: tRNA Bound to the Aggregates of 0.5 mM POPC Vesicles (Diameter ∼ 160 nm) Containing Varying Fractions of CTAB molar fraction of CTAB in POPC (%)a
[CTAB] (µM)b
[RNA] (µM)c
[RNA] of nucleotides/charges (µM)d
2.0 3.5 5.0 10.0
10.0 17.5 25.0 50.0
0.091 0.195 0.290 0.576
6.83 14.63 21.75 43.20
a Molar fraction of CTAB in 0.5 mM POPC. b Molar concentration of CTAB in POPC. c Molar concentration of bound tRNA (calculated from absorption at λ ) 260 nm). d Concentration of nucleotides/charges as obtained by multiplication of [RNA]c with constant factor 75 (number of nucleotides). This concentration equals the concentration of nucleotides as well as the concentration of charges (each nucleotide providing one negative charge).
mM) into the vesicles. Vesicle coaselscence is generally accompanied by a leakage of the vesicle interor,13 and thus a loss of calcein would indicate that processes beyond simple aggregation were involved. Encapsulation of high calcein concentrations inside vesicles leads to a weak fluorescence signal because of the effect of self-quenching.14 Content leakage of vesicle pools gives rise to an increase of the signal, which is caused by the dilution of the fluorophor in the external aqueous medium and the diminishing of the self-quenching effect. In our experiment, however, there was no increase of the fluoresence signal after a cycle of aggregation and disaggregation, indicating that the processes did not involve vesicle coalescence. Consequently, we conclude that the vesicles undergo simple aggregation, as not expected elsewise, taking into account the complete reversibility of the process upon addition of RNase A. To investigate the amount of tRNA bound to the aggregates, we prepared large vesicles with varying molar fractions of CTAB (ranging from 2 to 10%). After addition of tRNA, the aggregates were centrifuged and dissolved in sodium cholate, and the tRNA concentration was measured by UV/vis spectroscopy at 260 nm (see Material and Methods). A linear relation was obtained between the tRNA bound to the vesicles and the molar fraction of CTAB, which suggests that the aggregation is probably mainly driven by electrostatic interaction (Figure 4). In addition, the data presented in Table 1 show that the charge-corrected concentration of tRNA (concentration of nucleotides and negative charges, see Materials and Methods, UV/Vis Spectroscopy) and the concentration of CTAB for different molar fractions of CTAB in the POPC matrix are close to each other. This suggests that the aggregation processes should mainly be due to charge neutralization.
Figure 5. Light microscopy of aggregates from large (∼160 nm diameter) 0.5 mM POPC/3.5% CTAB vesicles upon addition of tRNA (final concentration 1.2 µM) injected from tRNA stock solution in 20 mM sodium phosphate buffer (pH 7.0). Bar represents 25 µm.
The ratio between tRNA and vesicles in the case of 3.5% molar fraction of CTAB corresponds to ca. 85 tRNA molecules bound per vesicle (assuming monodisperse vesicles of diameter 160 nm). In Figure 5, a result from light microscopy studies is shown. It can be seen that amorphous aggregates have been formed. The question that came to mind at this point concerns the possible physical properties of the vesicles that determine the aggregation phenomenon. Interesting at this regard is the effect brought about by the addition of cholesterol. In fact, POPC/ 3.5% CTAB vesicles that contain 20% molar fraction of cholesterol did not show any aggregation upon addition of tRNA. It is well-known that phosphatidylcholine membranes that acquire cholesterol become stiffer than membranes composed of pure phosphatidylcholine.15,16 It is therefore reasonable to assume that the bilayer flexibility may be an important factor for membrane aggregation. The argument of the possible relevance of stiffness for the vesicle aggregation phenomena will become more evident after consideration of the next series of experiments concerning the small vesicles. III. Stability of Small POPC/CTAB Vesicles. Let us now consider the case of small vesicles of the same composition (POPC/3.5% CTAB) that were only one-half smaller (∼80 nm diameter) but showed a quite different behavior. Figure 1, as already mentioned, shows that CTAB is efficiently uptaken also by the smaller vesicles, and therefore the small vesicles own a comparable charge density. As a consequence, one would expect the same kind of interaction with tRNA as for the large vesicles. Under otherwise the same conditions, however, these vesicles did not show any tendency to aggregate upon addition of tRNA (Figure 2) even if the tRNA concentration was increased by 1 order of magnitude. The stability of small vesicles held true for CTAB molar fractions up to ca. 5.5%; at 3.5% CTAB molar fraction, there was also no aggregation observed when the concentration of POPC was doubled to 1 mM. Consequently, rather the size of the vesicles and not their concentration was the critical parameter for aggregation. Notice that the concentration of surfactant for small and large vesicles was the same in both cases, which means that also the total surface area is about the same. This also means that the number of small vesicles exceeds the number of the large ones by a factor of 4.4.8 The difference in aggregation behavior between small and large vesicles was also obtained when CTAB was replaced by the double-chained cationic surfactant DDAB (didodecyldimethylammonium bromide).
RNA Selectively Interacts with Vesicles
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Figure 6. Zeta potential for small vesicles (∼80 nm diameter) 0.5 mM from POPC/3.5% CTAB upon addition of tRNA in 20 mM sodium phosphate buffer (pH 7.0), applied voltage 150 mV, sensed voltage ca. 130 mV.
The difference between the two classes of vesicles held true within CTAB molar fractions from ca. 2 to ca. 5.5% of the total lipid. Above 5.5% CTAB molar fraction, the small vesicles aggregated in the presence of tRNA, but the aggregation was much less pronounced than in the case of the large vesicles. Small vesicles (containing, for example, 5% CTAB) did not aggregate even though their zeta potential was higher than that of large vesicles containing, for example, 2.5% CTAB that aggregated. On the other hand, our data point to a large difference between the two classes of vesicles upon addition of tRNA. The question that arises at this point is whether tRNA binds or not to the small vesicles. Detection of bound tRNA was achieved by applying fluorescence and zeta potential measurements. In the fluorescence assay, the sample containing 0.5 mM small vesicles from POPC/3.5% CTAB and tRNA was fractionated by spin column chromatography, and the quantification of bound tRNA was achieved by addition of fluorophor Ribogreen (see Materials and Methods, Spin Column Chromatography and Fluorescence Measurements). It was found that tRNA in fact binds to the small vesicles and that the concentration of bound tRNA is about 15-20 nM corresponding to 1.3-1.5 µM nucleotide charges (data not shown). Since the concentration of CTAB is 17.5 µM (coresponding to 3.5% CTAB in 0.5 mM POPC), this means a ratio of ca. 12 between CTAB molecules and nucleotides. In the electrokinetic assay, tRNA solution was stepwise added to 0.5 mM POPC/3.5% CTAB vesicles. As shown in Figure 6, a clear decrease of the zeta potential was detected, indicating a decrease of the net charge because of the binding of tRNA to the vesicle surface. In particular, as can be seen in Figure 6, the addition of tRNA operated a decrease of zeta potential until the concentration of tRNA in the suspension was ca. 20 nM. Further addition of tRNA solution did not give rise to any further decrease of the signal. This value can be considered as a saturated state of the vesicle surface. Summarizing, small vesicles are also capable of binding tRNA, but the binding does not trigger an aggregation process of the vesicles. IV. Selective Aggregation. The discriminating capability of the tRNA molecule can be understood as a selection mechanism and the question at this point was whether such a selection is also operating in a sample in which both populations of vesicles are present. To clarify this question, both populations (with a CTAB molar fraction of 3.5%) were mixed in an equimolar ratio, and the aggregation process was followed by DLS (Figure 7 A-C) and by the OD increase (Figure 8). In Figure 7, it can be seen that at the beginning there is a broad peak of the mixed vesicle population because DLS cannot resolve under our conditions the close maxima of the two size distributions. The addition of tRNA operates a discrimination between the two
Figure 7. Vesicle size selection induced by tRNA binding. tRNA (total concentration 1.2 µM) is added to an equimolar mixture of small (79.6 ( 0.8 nm diameter) and large vesicles (159.4 ( 3.3 nm) from 0.5 mM POPC/3.5% CTAB in 20 mM sodium phosphate buffer (pH 7.0) followed by DLS measurement. (A) Shows the initial size distribution of the 1:1-mixture, (B) the size distribution after 4 min of the aggregation process, (C) the stable final size distribution after 15 min with one peak at small diameters (∼90 nm) and a second peak corresponding to the aggregated state of the large vesicles (>1000 nm).
Figure 8. Aggregation of small (79.6 ( 0.8 nm diameter) and large 0.5 mM POPC/3.5% CTAB vesicles (159.4 ( 3.3 nm) followed by OD measurement, λ ) 400 nm, molar ratio (between small and large vesicles) 1:1, final concentration of tRNA 1.2 µM in 20 mM sodium phosphate buffer (pH 7.0).
populations in the sense that it leads to a shift of one peak to values of larger radii under retention of a narrowed peak in the region of small radii. These data show that the mixture has been split in fractions of aggregating and nonaggregating vesicles. To further verify this result, centrifugation was applied to the sample. The
14548 J. Phys. Chem. B, Vol. 109, No. 30, 2005
Figure 9. Schematic illustration of the selective aggregation occurring in a sample containing small (∼80 nm diameter) and large vesicles (∼160 nm diameter) from POPC/3.5% CTAB. For an equimolar ratio of the two populations, there is a 4.4-fold excess of the small vesicles in terms of particle number. For the sake of simplicity, the populations in the illustration are represented as monodisperse species.
Thomas and Luisi given in terms of relative intensity, which underestimates the smaller vesicles. The size threshold for aggregation was also estimated in another experiment in which the size and size distribution of the small vesicles from POPC/3.5% CTAB in the presence of tRNA were followed in a long-term experiment. We reported about the swelling of differently sized vesicles,17 and because of the natural swelling of POPC/3.5% CTAB vesicles, their mean diameter approaches bit by bit the critical size. In fact, after several days, the DLS showed the emergence of a second peak indicating the formation of aggregates, which did not happen if either CTAB or tRNA were missing in the sample (control experiments). The information from this experiment is that there is in fact aggregation also for the former small vesicles as soon as they grow to ca. 110-120 nm. Nevertheless, it should be emphasized that the observations made to a certain extent depend on the quality of the vesicle size distributions that request a high narrowness particularly for the small vesicles. Concluding Remarks
Figure 10. Vesicle size selection by tRNA (final concentration 1.2 µM) added to 0.5 mM POPC/3.5% CTAB vesicles obtained by extrusion through 400-, 200,- 100-, and 50-nm pore diameter (ratio 1:1:1:1) in 20 mM sodium phosphate buffer (pH 7.0). The broad peak (-9-) corresponds to the size distribution of the mixed population after the extrusion process. The sharp peak (-b-) centered around 100 nm corresponds to the size distribution of the nonaggregated fraction, which remains in the supernatant after centrifugation (98.2 ( 3.4 nm). After resuspension of the aggregates in buffer containing 5 nM RNase A, the mean diameter is 182.8 ( 4.0 nm (-2-).
supernatant showed a size distribution and a mean vesicle diameter of 89 nm and thus was close to that of the small vesicle population (∼80 nm). The resuspended pellet (after disaggregation by RNase A) contained the large vesicles with a characteristic diameter of ca. 165 nm (data not shown). All taken together, these results show that tRNA is capable of discriminating between vesicles of different sizes in separated samples as well as in a mixed sample containing both populations. A pictorial illustration of the different behavior between large and small vesicles is given in Figure 9, where the large vesicles aggregate in a finite assembly state and the small ones do not. The illustration also takes case of the fact that an equimolar ratio of the two populations corresponds to a 4.4-fold excess of the small vesicles in terms of particle number.8 V. Determination of the Size Threshold. Being clear that the size and the size distribution of the vesicles operated the selection, it was interesting to estimate the size threshold at which this discrimination occurred. To this aim, we prepared a mixed vesicular population of POPC/3.5% CTAB vesicles by extrusion through 400-, 200-, 100-, and 50-nm pores size in a 1:1:1:1 molar mixing ratio. This mixture gives a very broad peak in the DLS size distribution with average diameter 154 nm, approximating a continuum of vesicle sizes (Figure 10). After addition of tRNA, centrifugation, and separation, the supernatant and the resuspended pellet were analyzed by DLS and it was found that the nonaggregated fraction had a mean diameter of 98 nm and that the aggregated fraction (after digestion by RNase A) was 183 nm in diameter. This means that the critical size that induces aggregation has an average diameter in the range of ca. 100 nm. Notice that Figure 10 is
The aim of our work was to demonstrate a possible interaction between the molecular classes of RNA and vesicles. To restrict the investigation range, we have focused attention on whether RNA might discriminate between vesicles of different sizes. Such an effect is in general interesting from the perspective of biophysical interactions, and in particular one could bring the argument that this discrimination might have been of importance for prebiotic chemistry. We have shown that such a size selection is in fact attainable under favorable experimental conditions. The vesicle mean diameter difference between the two vesicle size distributions is only a factor of 2, which indicates that the selection is indeed fine-tuned. At this point, several interesting questions open up. One is about the physical mechanism responsible for such a selection. It appears at first sight that in our system mostly electrostatic interactions are operative. If this were the only factor, one would not see why there should be a difference between the two vesicle populations (each containing 3.5% CTAB) since the positive charge density (because of CTAB units) is comparable. As mentioned above, a preparation of large vesicles containing 2.5% CTAB started aggregating even if its zeta potential was lower than the potential of a preparation of small vesicles, containing as much as 5% CTAB. Furthermore, the charge density in the latter case was higher, but nevertheless these vesicles did not undergo aggregation. Clearly, then, physical factors arising from the vesicle size must be responsible for the observed difference, for example, because of effects brought about by the curvature radius and the bilayer flexibility. In fact, the substitution of 20% POPC by the stiff surfactant cholesterol completely prevented the large vesicles from aggregation. On the other hand, it is usually accepted that smaller vesicles with average diameter of around 50-80 nm are particularly stable, whereas larger ones tend to aggregate more rapidly and are therefore more reactive. It has been shown, for example, that larger vesicles tend to grow faster upon addition of fresh surfactant than smaller ones.18 Consequently, it should be taken into consideration that the aggregation of the larger vesicles with tRNA might be due to their enhanced reactivity. The other important question to which it is even more difficult to give an answer is whether the observed selectivity of tRNA toward vesicles of a certain size and thus toward a certain physical property may have been relevant for the following steps of implementing an increase of molecular complexity and
RNA Selectively Interacts with Vesicles specificity as a way to the first cells. A possible scenario for RNA cells has been recently presented.19 Materials and Methods Chemicals. tRNA (E. coli, Type XX) and RNase A (Type II A) were purchased from Sigma-Aldrich, Steinheim, Germany. tRNAPhe (E. coli) was from Boehringer Mannheim, Germany. CTAB (cetyltrimethylammonium bromide), DDAB (didodecyldimethylammonium bromide), sodium dihydrogen phosphate dihydrate, calcein disodium salt, and cholic acid sodium salt were from Fluka, (Buchs, Switzerland), sodium hydroxide from Synopharm, (Schweizerhall, Switzerland), and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) was from Avanti Polar Lipids (Alabaster, AL). Cholesterol was purchased from Cosmo SpA - Lainate. Ribogreen was from Molecular Probes (Leiden, Netherlands), and gel Sepharose 4B was from Amersham Pharmacia Biotech AB (Uppsala, Sweden). All aqueous solutions were prepared in fresh deionized water, obtained by a Milli-RX20 apparatus from Millipore. Optical Density Measurements. Optical density measurements were carried out at 25.0 °C recording the absorbance at 400 nm with a Hewlett-Packard 8452a diode array spectrophotometer. Quartz cells with path length of 1 cm were used, and the sample volume was chosen to 1 mL. In all experiments, the tRNA was added from concentrated stock solution (solved in sodium phosphate buffer pH 7.0) into the vesicular suspension giving a final concentration of 1.2 µM. In terms of nucleotide charges, this concentration corresponds to an excess of RNA (90 µM in nucleotides/charges, as tRNA has 75 units) with respect to the CTAB in the POPC vesicles (max molar fraction 10% respectively 50 µM). UV/Vis Spectroscopy. Quantification of the RNA bound to the aggregates was done with a Hewlett-Packard 8452a diode array spectrophotometer at 260 nm. After aggregation of the vesicles induced by addition of tRNA, the aggregates were precipitated by centrifugation with an ALC microcentrifuge (model 4214) at 10 000 rot/min for 10 min. The supernatant was separated from the pellet and the latter one was resuspended in buffer containing 200 mM sodium cholate. The obtained A260 values were converted into tRNA molar concentration according to eq 1, A260 ) 1.6 µM tRNA. The values for charge-corrected concentration of tRNA were obtained by multiplication of the tRNA molar concentration with factor 75 (each tRNA molecule providing 75 negative charges). Dynamic Light-Scattering Measurements. Determination of vesicle size (hydrodynamic diameter) and size distribution was done by DLS (dynamic light scattering) with a ZetaSizer 5000 (λ ) 633 nm) at 25.0 °C. Measurements and analysis were performed at scattering angle 90°. Size distributions are reported in terms of relative intensity, that is, large particles are overestimated. Zeta Potential Measurements. Zeta Potential of POPC/ CTAB vesicles was measured with a ZetaSizer 5000 (λ ) 633 nm) at 25.0 °C. Analysis was performed with a fifth order polynomial fit (modeling the behavior of the background part of the correlation function). Column Chromatography and Fluorescence Measurements. Spin column chromatography for tRNA quantification was performed at room temperature with 1-mL volume spin columns containing gel Sepharose 4B. Applied sample volume was 100 µL, and fractionalization was done with an ALC 4236A centrifuge at 2000 rpm with each collection step lasting 2 min. Collected fraction volumes ranged from 80 to 125 µL. Detection was done directly with a Perkin-Elmer luminescence
J. Phys. Chem. B, Vol. 109, No. 30, 2005 14549 spectrometer LS 50 B, thermostated at 25 °C (with a Neslab RTE-200 thermostate). The vesicles were destroyed with different volumes of 200 mM cholate, dependent on the fraction volume, giving a constant cholate concentration of 25 mM. Ribogreen was added to the samples, giving a complex with fluorescence emission maximum at 525 nm. Excitation wavelength was 480 nm. Calcein entrapment was performed by film resuspension in 10 mM calcein/phosphate buffer solution followed by column chromatography at room temperature with a column of ca. 10 mL volume containing gel Sepharose 4B. The applied sample volume was 150 µL. Fractions of ca. 500 µL volumes were collected. Detection of vesicle calcein release upon tRNA addition was done with a Perkin-Elmer luminescence spectrometer LS 50 B, thermostated at 25 °C (with a Neslab RTE-200 thermostate). Excitation wavelength was chosen to 488 nm, and the emission maximum was 513 nm. For verifying successful incorporation of calcein, the vesicles were finally destroyed by addition of a 40-fold excess of cholate. Light Microscopy. Light microscopy was performed with a phase-contrast Axiovert 135 inverted light microscope (Zeiss AG, Germany), equipped with a 40× long working distance objective lens (LD Achroplan Ph2) and an additional 1.6× magnification ring. POPC/CTAB Vesicles. Method i. POPC/CTAB vesicles were prepared by applying the film method. Solid POPC and CTAB were dissolved in CHCl3 giving 75 mM concentrated stock solutions. According to the molar fraction of CTAB in POPC, both solutions were mixed in a round-bottomed flask followed by removing the solvent by evaporation (Bu¨chi Rotavapor) and high vacuum-drying of the film overnight. The film was then dispersed by vigorous shaking and short sonication in 20 mM sodium phosphate buffer (pH 7.0) giving a 10 mM suspension. Ten cycles of freezing and thawing were applied to the samples using liquid nitrogen for reducing the lamellarity of the vesicles.20 Method ii. The film method was applied for POPC as described under method i. After dispersion of the film in buffer and freezing and thawing, CTAB was introduced subsequently from 10 mM solution. In both methods, the suspensions were then extruded (Extruder, Lipex Biomembranes, Vancouver, Canada) through two stacked polycarbonate filters (Nuclepore, Sterico AG, Dietikon, Switzerland) starting with 400-nm pore diameter followed by 200, 100, and 50 nm depending on the experimental setup; each extrusion step was repeated 10 times. Afterward, the suspensions were diluted with buffer from 10 mM to final concentrations of 0.5 mM, respectively 1 mM. Method iii. The film method was applied for POPC as described under method ii. The film was dispersed in the buffer followed by freezing and thawing of the sample. The pure POPC vesicles were then extruded to the final size and CTAB was added subsequently. Vesicles prepared by method iii were used only in experiments described under section I. Acknowledgment. Pasquale Stano is acknowledged for critical reading of the article. References and Notes (1) Khvorova, A.; Kwak, Y.-G.; Tamkun, M.; Majerfeld, I.; Yarus, M. Proc. Natl. Acad. Sci. U.S.A 1999, 96, 10649-10654. (2) Vlassov, A.; Yarus, M. Mol. Biol. 2002, 36, 389-393. (3) Vlassov, A.; Khvorova, A.; Yarus, M. Proc. Natl. Acad. Sci. U.S.A 2001, 98, 7706-7711. (4) Janas, T.; Yarus, M. RNA 2003, 9, 1353-1361.
14550 J. Phys. Chem. B, Vol. 109, No. 30, 2005 (5) Albi, E.; Micheli, M.; Viola Magni, M. P. Cell Biol. Int. 1996, 20, 407-412. (6) Chen, I. A.; Roberts, R. W.; Szostak, J. W. Science 2004, 305, 1474-1476. (7) Hunter, D. G.; Frisken, B. J. Biophys. J. 1998, 74, 2996-3002. (8) For this calculation, aggregation numbers of 50 000 surfactant molecules per small vesicle and 220 000 molecules per large vesicle were assumed. In turn, these figures have been calculated on the basis of a crosssectional area of POPC of 0.72 nm2 and a bilayer thickness of 3.7 nm, neglecting the surfactant packing parameters of CTAB. Lonchin, S.; Luisi, P. L.; Walde, P. Phys. Chem. B 1999, 103, 10910. (9) Cevc, G. Chem. Phys. Lipids 1993, 64, 163-186. (10) Yaroslavov, A. A.; Udalyk, O. Y.; Kabanov, V. A.; Menger, F. M. Chem. Eur. J. 1997, 3, 690-695. (11) Roy, M. T.; Gallardo, M.; Estelrich, J. J. Colloid Interface Sci. 1998, 206, 514. (12) Wiersema, P. H.; Loeb, A. L.; Overbeck, J. T. G. J. Colloid Interface Sci. 1966, 22, 78.
Thomas and Luisi (13) Boni, L. T.; Batenjany, M. M.; Neville, M. E.; Guo, Y.; Xu, L.; Wu, F.; Mason, J. T.; Robb, R. J.; Popescu, M. C. Biochim. Biophys. Acta 2001, 1514, 127-138. (14) Hamann, S.; Kiilgaard, J. F.; Litman, T.; Alvarez-Leefmans, F. J.; Winther, B. R.; Zeuthen, T. J. Fluoresc. 2002, 12, 139-145. (15) Liang, X.; Mao, G.; Ng, K. Y. S. J. Colloid Interface Sci. 2004, 278, 53-62. (16) Liu, D.-Z.; Chen, W.-Y.; Tasi, L.-M.; Yang, S.-P. Colloids Surf., A 2000, 172, 57-67. (17) Thomas, C. F.; Luisi, P. L. J. Phys. Chem. B 2004, 108, 1128511290. (18) Cheng, Z.; Luisi, P. L. J. Phys. Chem. B 2003, 107, 11940-11945. (19) Szostak, J. W.; Bartel, D. P.; Luisi, P. L. Nature 2001, 409, 387390. (20) MacDonald, R. C.; MacDonald, R. I. Applications of freezing and thawing in liposome research. In Liposome Technology-; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1993; Vol. 1, Chapter 13, pp 209223.
