Soft Matter PAPER. ssrna base pairing at a bilayer interface can be controlled by the acyl chain order. Dynamic Article Links C <

Transcription

1 Soft Matter / Journal Homepage / Table of Contents for this issue Dynamic Article Links C < Cite this: Soft Matter, 2012, 8, ssrna base pairing at a bilayer interface can be controlled by the acyl chain order Agnes Michanek,* Mathias Bj orklund, Tommy Nylander and Emma Sparr PAPER Received 8th September 2011, Accepted 1st August 2012 DOI: /c2sm06700e RNA lipid interactions are central to structure and function in biological systems as well as to the development of new applications in medicine and biotechnology. We have studied adsorption and base pairing of short RNA oligonucleotides at model lipid membranes with different compositions by means of QCM-D, confocal microscopy and ITC. The major finding is that base pairing of short complementary RNA strands can be controlled by the acyl-chain chain order, i.e. chains in the solid vs. liquid state, in a deposited bilayer. It was shown that the base pairing with a complementary strand ssrna takes place at the bilayer when the first strand is pre-adsorbed to a bilayer with solid chains, but not when the first strand is pre-adsorbed to a liquid crystalline bilayer with fluid chains. The results imply that the ssrna hydrophobic bases are not accessible to the complementary strand bases when RNA is adsorbed to a fluid bilayer, which can be due to hydrophobic interactions with the apolar layer in the fluid bilayer. It is also likely that the difference in lipid phase behaviour affects the kinetics for the base pair reaction at the surface. The corresponding base pairing experiment at the interface of a soft cationic polymer layer consisting of poly(amido amine) PAMAM dendrimers of generation 4 yielded similar results. RNA and DNA were found to adsorb to mixed bilayers that contain the naturally occurring cationic lipid sphingosine as well as to bilayers that contain more conventional surfactants, and it is found that the surface with adsorbed nucleic acid is close to electroneutral at saturation. Introduction It has been shown that short RNA oligomers have important regulatory functions in the cell. One such example is the mirnas (micro RNAs), which are non-coding short ssrna sequences containing bases. These small RNA oligomers are believed to be important for up and down regulation of cellular processes such as cell death and cell proliferation 1 as well as for the initiation of several types of cancer. 2,3 Thus it is important to be able to control mirna activity, as this can lead to the development of new treatment strategies, 4 for example anti-cancer drugs that use mirna. 5 Of particular relevance for our study are strategies that address the challenge of delivering mirna and sirna to the target inside the cell. Here, phospholipid-based drug delivery systems have been proven to have great potential. 6 9 Biophysical studies have shown that DNA molecules condense upon interaction with cationic amphiphiles A general problem related to the applicability of these model systems to, e.g., drug delivery is that most highly charged cationic (synthetic) surfactants are indeed toxic for living cells. 15 This motivates studies of biologically relevant cationic lipids, such as the sphingomyelin Division of Physical Chemistry, Center of Chemistry and Chemical Engineering, Lund University, P.O. Box 124, Lund, Sweden Electronic supplementary information (ESI) available. See DOI: /c2sm06700e derivative sphingosine. Sphingosine is a cationic single-chain amphiphilic sphingolipid, and one can therefore expect electrostatic attraction between this lipid and oppositely charged RNA or DNA. In model membrane systems, association of RNA and DNA to membranes composed of zwitterionic phospholipid and cationic sphingosine has previously been demonstrated. 16,17 RNA and DNA base-pairing is essential for control of cellular processes. The specific base-pairing reactions are also central to the design of nanostructures with pre-defined structure and function, which has applications in nanotechnology and biotechnology. The duplex formation of single RNA or DNA strands can be regulated by changing physical or chemical conditions, e.g., temperature, flow, micromanipulation, ionic strength or compacting agents In so-called DNA and RNA origami, the specificity in the interactions between the complementary bases enables construction of nanoscale objects. 24 Knowledge of how these interactions can be controlled by physical and chemical means enables rational design and construction of complex structures in the bulk and at surfaces. One beautiful example of the use of micro or rather nanometer manipulation to create designed structures is the use of an AFM tip to place DNA segments on different locations at a surface to create a specific pattern of DNA strands on mica surfaces. 25,26 An alternative approach would be to direct the duplex formation of DNA or RNA by changing the solution properties of the system. This requires a detailed characterization of how the nucleic acid Soft Matter, 2012, 8, This journal is ª The Royal Society of Chemistry 2012

2 duplex formation depends on the external conditions. It is known that duplex formation and helix unzipping can be controlled by changes in solution composition, 21,23 temperature 22 and enzymatic activity. 27 Another approach to control DNA and RNA assembly is to confine the base-pairing in nanopores 28 or on a solid surface or liquid interface that could be, e.g., a functionalized surface, 25 a mineral surface 29 or a lipid membrane. 30 The surface or the nanopore confinements then act as a scaffold to which the nucleic acid is attached or entrapped. In nature, RNA occurs both in double-stranded (ds) and single-stranded (ss) forms. For the single-stranded species, the apolar parts of the bases are likely prone to hydrophobic interaction with other species, e.g., the hydrophobic regions of a lipid membrane. The aim of the present study is to reveal how the adsorption of ssrna, and hence the base-pairing, can be regulated by changing the properties of a supported lipid bilayer. We use model systems composed of short RNA oligonucleotides and salmon sperm DNA together with mixed lipid bilayers composed of zwitterionic phosphatidylcholines (PC) as well as cationic sphingosine or cationic surfactants. We study base pairing reactions of different types of RNA that are adsorbed to lipid bilayers on a solid support. The idea is that the bilayer acts as a scaffold for assembly of RNA and that changing the properties of the bilayer can control the RNA base pairing reaction. We wish to explore whether changes in the bilayer properties can be used as an on/off switch for the base pairing reaction. One advantage of the chosen strategy is that the assembly is directed to a 2-D surface instead of the comparably large volume of a 3-D system, and therefore it generally requires less material. In particular, we compare the difference in nucleic acid adsorption to zwitterionic bilayers to that which occurs to mixed bilayers that include either cationic surfactant or the naturally occurring cationic lipid sphingosine. The association of nucleic acids in bulk and to the supported bilayers as well as to vesicles in bulk is studied by means of QCM-D, ITC and confocal microscopy. The base-pairing by sequentially adding complementary strands of ssrna was also monitored by these techniques. The corresponding base pairing experiment was also conducted at the interface of a soft cationic polymer layer consisting of poly- (amido amine) PAMAM dendrimers of generation 4. Material and methods DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine), D-erythrosphingosine (d18 : 1), DODAB (dioctadecyl-dimethyl-ammonium bromide), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rh-PE), and DOTAP (18 : 1 TAP, 1,2-dioleoyl-3-trimethylammonium-propane) with 99% purity were purchased from Avanti Polar Lipids, Inc. (Alabaster, USA). Chloroform and methanol (both 99.8% purity) were purchased from Merck (Darmstadt, Germany). HEPES and EDTA $ 99.5% purity were purchased from Sigma Aldrich (Missouri, USA). GelStarÒ nucleic acid gel stain was purchased from Lonza (Rockland, ME, USA). The poly(amido amine) PAMAM dendrimers of generation 4 with ethylenediamine cores were purchased from Sigma. The PAMAM dendrimers were dissolved in methanol and were dried overnight in a vacuum oven prior to use and then left to hydrate at 4 C for 1 2 days. The short ssrnas used were ssrna 10-polyA (polya 10 bases), ssrna 10-polyU (polyu 10 bases), ssrna 10-s1 ( UCUUCUA CUU, 10 bp) and ssrna 10-s2 ( AAGUAGAAGA, 10 bp). These were synthesized and HPLC purified by MWG Biotech (Ebersberg, Germany). The ssrna 10-s1 and ssrna 10-s2 were designed as complementary strands for duplex formation of a short dsrna ( UCUUCUACUU, 10 bp), where the complementary strands assemble without mismatching and selfassociating upon mixing (design kindly provided by Prof. Luc Jaeger, UCSB, USA). Double stranded salmon sperm DNA, dsdna 2000 ( bp), was purchased from Invitrogen Life Technologies (California, USA). trna (60 90 bases) was purchased from Sigma-Aldrich (Missouri, USA). The RNA samples were analyzed using ICP MS (inductively coupled plasma mass spectrometry) to determine the RNA concentration (i.e. the concentration of phosphorus), the counterion (Na + ) concentration and the content of divalent ions (i.e. calcium, magnesium, iron, aluminum and copper) of each sample. The analysis showed that the samples contained very low amounts of divalent ions (less than mol Ca 2+ per mol RNA and even lower for the other multivalent ions). The counterion concentrations were determined to be ca. 4 mol Na + per mol RNA. One of the batches of ssrna (the complementary strand with sequence AAGUAGAAGA) contained an insoluble contamination, which was removed with ultracentrifugation, and the supernatant containing the RNA was collected. The supernatant did not contain any divalent ions. The RNA concentration in the supernatant was then determined by means of ICP MS. The same problem did not occur for the other RNA samples, which were used as received. All other chemicals were used as received without further purification. All solutions were prepared using ultrapure water from a Milli-Q Ultrapure water purification system from Millipore (Massachusetts, USA). All glassware was soaked in Hellmanex 2%, rinsed with de-ionized water, soaked in 10% hydrochloric acid for several hours and then rinsed 10 times with Milli-Q water. As RNA is sensitive to nuclease degradation, all glassware was sterilized at 200 C for 8 hours and all solutions were autoclaved (121 C for 20 minutes). Preparation of small unilamellar vesicles Stock solutions of the lipid mixtures used in the QCM-D and DSC studies were prepared by dissolving the lipids surfactants in chloroform/methanol 2 : 1. Smaller aliquots from the stock solutions were taken out and dried under a stream of air until the solvent evaporated and a thin lipid film remained. The lipid film was suspended in 10 mm HEPES 0.2 mm EDTA ph 7.0 and left above the lipid T m for at least 30 min before sonication. In order to deposit a bilayer on the SiO 2 coated QCM crystal, the vesicle fusion technique was used. 31 The lipid dispersion was sonicated using 30 second pulses at 30% amplitude (Vibracell tip sonicator, Sonics & Materials Ltd., USA) until a clear dispersion of small unilamellar vesicles (SUV s) was obtained, which usually takes less than 10 minutes. Care was taken not to overheat the sample. This journal is ª The Royal Society of Chemistry 2012 Soft Matter, 2012, 8,

3 Preparation of GUV s Giant unilamellar vesicles (GUVs) composed of DOPC Sph were prepared by electroformation 32,33 in a chamber with Pt electrodes. 34 Lipids in chloroform/meoh 2 : 1 (v/v) (0.2 mg ml 1 ) were deposited on the Pt electrodes using a Hamilton syringe and the solvent evaporated in a vacuum chamber for at least 2 h. After adding 200 ml of the HEPES buffer into the chamber a peak-topeak voltage of 2 V and a frequency of 50 Hz were applied for 30 min, after which the voltage was increased to 6 V peak-to-peak for 2 h. The fluorescent lipid analogue was added at a concentration of 1 mol% of the total amount of lipids. QCM-D Quartz crystal microbalance with dissipation (QCM-D) measurements were performed using a Q Sense E4 system from Q Sense (Gothenburg, Sweden) with four temperature controlled flow modules. One sensor crystal was placed in each flow module, and the crystals used for the experiments were quartz crystals with a fundamental frequency of 4.95 MHz covered by a thin gold surface connected to the two electrodes. The gold surface was then coated with a 50 nm thick SiO 2 layer (QSX 303, Q Sense). An external peristaltic pump (Ismatec IPC-N 4) ensured a constant flow of liquid through the modules. Prior to use, the sensor crystals were cleaned in 2% SDS solution for a minimum of 1 hour, washed with MQ water, rinsed with ethanol, dried under nitrogen and thereafter treated in a plasma cleaner from Harrick Scientific (New York, USA) for 5 minutes in reduced air plasma at a pressure of 0.02 mbar. Directly after plasma cleaning the crystals were inserted into the QCM-D cells. Before each measurement, the crystals were allowed to equilibrate in water until a stable baseline was reached. The QCM crystals were then equilibrated with the 10 mm HEPES 0.2 mm EDTA buffer prior to deposition of the bilayer. Deposition of the bilayer is done by pumping the lipid SUV dispersion through the cell at a pump speed of 100 ml min 1. The formation of a stable bilayer is fast and even after 3 5 minutes it is possible to start rinsing the surfaces with buffer. The bilayers formed usually have a close to 100% surface coverage that corresponds well to the coverage achieved using the detergent dilution technique. 41 The nucleic acids were added continuously at a flow rate of 10 ml min 1 ; nucleic acid concentration was varied in the different experiments. In order for the nucleic acids to reach the cell faster, the pump speed during the first 1 min and 15 s was 100 ml min 1,as the lag time otherwise becomes very long. All experiments were performed at 20 or 25 C. The wet mass of the lipid film adsorbed to the silica was calculated according to the Sauerbrey expression Dm ¼ C O n Df (1) where C z 17.7 ng Hz 1 cm 2 for a 5 MHz crystal and o n is the overtone number. 35 The validity of applying this equation to non-rigid biomolecular systems has been addressed by H o ok et al., 36 who concluded that the Sauerbrey expression is a good approximation for thin and homogeneous adsorbed layers. However, an acoustically rigid film may also trap solvent, which becomes sensed as an additional mass. Hence, only in cases when the amount of coupled water is low does the Sauerbrey mass correspond well to the adsorbed mass. Planar supported lipid bilayers constitute one such example, as verified with other methods such as ellipsometry. 37 In the case of polynucleotide coupling to supported lipid bilayers, the situation is more complicated, 38 but as the damping is low, relative differences can still be trusted with high accuracy. Justified by the fact the adsorption of the DNA and ssrna was observed to cause only minor change in the dissipation, and since we are mainly concerned with relative differences in the amount of polynucleotides adsorbed to phospholipid bilayers with different lipid composition, we applied the Sauerbrey expression to quantify the adsorbed mass of ssrna and DNA. Previous analyses of DNA adsorbed to phospholipid bilayers have shown good agreement for the adsorbed amounts of DNA as measured by ellipsometry and QCM-D with Sauerbrey analysis. 39 Confocal microscopy Confocal microscopy measurements were performed using a Zeiss LSM 510 Meta inverted microscope. The green and red fluorescence signals were acquired using double excitation (488 nm from an Argon/2 laser and 561 nm from a DPSS laser) and detection (BP and LP 575). The GUVs were inspected by microscopy before the addition of the ssrna and GelStarÒ. ssrna and GelStarÒ samples were added to final concentrations of 76 mm. DSC A vpdsc differential scanning calorimeter (MicroCal, Inc., North Hampton, USA), with a cell volume of ml, was used to determine the T m for the mixed lipid surfactant samples in order to determine what phase they were present in under the experimental conditions used during the QCM-D measurements. Multi-lamellar vesicle samples with a lipid concentration of 1 mm of the different lipid surfactant mixtures were prepared and the chain melting transition was monitored by increasing the temperature in the measuring cell by 1 C min 1 scanning over a temperature interval from 5 to 60 C. ITC The enthalpy of interaction was measured using a vpitc isothermal titration calorimeter (MicroCal, Inc., North Hampton, USA) with an active cell volume of 1.44 ml. The syringe was filled with a solution containing ssrna 10-s1 strands with a concentration of 0.2 mg ml 1 and the cell was filled with a solution containing the complementary strand ssrna 10-s2 with a concentration of 0.01 mg ml 1. Experiments were performed at 20 C, below the denaturation temperature of the dsrna 10 complex (that has a melting temperature of 26 C as stated by the manufacturer). The injection size was 10 ml and the injection speed was set to 0.5 mls 1. The total number of injections was 29 and the stirring speed was 300 rpm. A 2 ml injection was done prior to the ml injections to assure that the syringe concentration was correct as there may be a small dilution of the solution at the tip of the injection syringe as the device is put into place in the injection cell Soft Matter, 2012, 8, This journal is ª The Royal Society of Chemistry 2012

4 Results We explore ssrna association and duplex formation at lipid bilayers, and how this is affected by the bilayer properties. One key question is how ssrna associates to the bilayer surface, if it orients with the charged backbone toward the headgroups of the oppositely charged lipids with the hydrophobic bases protruding into the solution, or if the hydrophobic bases are more buried in the hydrophobic region of the bilayer. In previous studies, we have shown penetration of ssrna 10-polyA into liquid expanded monolayers where the hydrophobic hydrocarbon chains of the lipids are exposed, while there is no penetration into liquid condensed monolayers with ordered chains. 20 For the latter case, adsorption of ssrna 10-polyA was detected as a layer just beneath the lipid headgroup, which was also the case for dsdna 2000 both at liquid expanded and liquid condensed monolayers. One can expect analogous behaviour for ssrna 10-polyA and dsdna 2000 on fluid lipid bilayers, and this would in turn affect the accessibility of the ssrna 10-polyA from the bulk solution. In this paper, we study the base-pairing of two complementary ssrna 10 strands at a lipid bilayer with different acyl-chain packing. For this purpose ssrna strands are adsorbed at the deposited bilayer, and then the complementary ssrna strands are added sequentially. A doubling of the adsorbed mass then suggests that base-pairing takes place. To rule out that the increase is a consequence of non-specific binding, the corresponding experiment was also performed with ssrna that have a non-complementary sequence. This experiment is performed for lipid bilayers with different properties (charge and phase behaviour). The analysis of the base-pairing experiments relies on thorough characterization of ssrna association to lipid bilayers with different properties. The aim was to find conditions where the lipid bilayer can be saturated with ssrna, and where the ssrna does not desorb upon dilution with buffer. In the first part of this paper, we describe experimental studies of ssrna adsorption to mixed zwitterionic cationic bilayers. We discuss bilayer systems with varying charge density, and systems including different cationic lipids. We also describe studies of ssrna adsorption to different mixed lipid systems that form either a fluid bilayer (bilayer with fluid chains) or a solid bilayer (bilayer with solid chains). Thereafter, we describe studies of ssrna base-pairing for the chosen model system in bulk solution. Finally, the results from RNA base-paring experiments at bilayers with different properties will be discussed. Deposition of lipid bilayers Supported lipid bilayers were prepared using the vesicle fusion technique. 40 This resulted in close to full surface coverage with deposited amount of lipids of ng cm 2 for the fluid bilayers and ng cm 2 for the solid bilayers. From the experiments with varying amounts of charged lipid surfactant in the fluid bilayer, we further conclude that the mean area per lipid acyl-chain in each leaflet of the lipid bilayer is virtually not affected by the charge density under the conditions used in this study. This is illustrated in Fig. 1 for mixed DOPC DOTAP and DOPC sphingosine bilayers. The figure further shows that the mean area per acyl-chain is roughly the same for DOTAP and for sphingosine containing bilayers. It is Fig. 1 Area per acyl-chain for DOPC DOTAP and DOPC sphingosine mixtures with different composition. The area per acyl chain is roughly the same independent of the amount of bilayer charge density. The percentage of charged chains refers to the number of headgroup charges averaged over the number of chains. T ¼ 25 C. important to note that we have chosen to compare the DOTAP and the sphingosine containing bilayers using the percentage of chains belonging to the charged lipids in the bilayer, as DOTAP has two acyl-chains and sphingosine has only one acyl-chain. The figure includes data from several measurements for bilayers with different compositions, and the deviation in area per chain is relatively small in-between the different measurements both for DOTAP and for sphingosine. The adsorbed amount of lipid at the SiO 2 -coated QCM crystal and the change in dissipation corresponds well to previous reports for deposited lipid bilayers in similar systems. 41 Finally, we observed that it was generally more difficult to deposit bilayers composed only of cationic lipids surfactants compared to bilayers composed of mixtures of zwitterionic and cationic lipids (Table 1). The deposition of bilayers composed of only cationic lipids surfactants generally resulted in lower surface coverage. The bilayer composition in all the experiments is given in mol%, for example the mixture DOPC sphingosine 50 : 50 contains 50 mol% DOPC and 50 mol% sphingosine. The composition refers to the average composition in the deposited bilayer, and does not take into account that the distribution of lipids between the upper and lower leaflet in the deposited bilayer might not be uniform, as has previously been described for other lipid systems. 42 For the present system, we expect that the content of cationic lipid is a bit higher in the leaflet that faces the slightly negatively charged silica surface compared to the average composition. Adsorption of DNA RNA saturation of the bilayer surface depends on the nucleic acid bulk concentration Fig. 2 shows typical data from a QCM-D experiment for the adsorption of ssrna 10-polyA to a deposited bilayer containing DOPC sphingosine 70 : 30. The raw data for the frequency change (Df) upon adsorption are recalculated to adsorbed mass using the Sauerbrey relation. First, the bilayer is deposited at the bare SiO 2 surface from the solution of SUV s (i), then the This journal is ª The Royal Society of Chemistry 2012 Soft Matter, 2012, 8,

5 Table 1 Adsorbed amount of ssrna 10 and dsdna 2000 to bilayers with different lipid composition and different phase behaviour. The percentage of charged chains refers to the number of headgroup charges averaged over the number of chains. T ¼ 25 C Lipid composition T m ( C) Phase Area per chain ( A) Nucleic acid Ads. amount (ng cm 2 ) Bases per lipid /+ DPPC Sph 50 : s 21 ssrna dsdna DOPC Sph 50 : 50 <0 L a 29 ssrna dsdna DOPC DOTAP 70 : 30 <0 L a 28 ssrna dsdna DPPC DOTAP 70 : L a 27 ssrna dsdna DOTAP 12 L a 35.6 ssrna DODAB Sph 85 : L a 33.1 ssrna Fig. 2 Experimental data obtained for the deposition of a DOPC sphingosine 70 : 30 (mol%) bilayer at the SiO 2 surface. The SUV s are added (i) followed by rinsing (ii) and then addition of ssrna 10-polyA (iii). Adsorbed mass (calculated from Df, Saurbrey eq., left axis) and change in dissipation (raw data DD, right axis). T ¼ 25 C. measurement cell is rinsed with buffer (ii), and then ssrna is adsorbed to the deposited bilayer (iii). In order to establish under which conditions the bilayer surfaces are saturated with ssrna, we studied the adsorption of ssrna 10-polyA to mixed DOPC DOTAP bilayers with composition 30 : 70 (i.e. 30 mol % DOPC and 70 mol% DOTAP) at varying ssrna 10-polyA bulk concentrations. The data for the adsorbed amount are summarized in Fig. 3 and S1 in the ESI. As a reference, the adsorption of dsdna 2000 to the lipid bilayers with the same composition was also studied. The adsorbed amount of both nucleic acids is presented as the number of negative charges from the nucleic acid bases per positive charge from the lipid headgroup ( /+) as a function of the nucleic acid concentration. In this way we are able to normalize the data with respect to small variations in bilayer coverage at the SiO 2 surface. Control experiments using ssrna 10 oligonucleotides with different sequences, including ssrna 10-polyA, ssrna 10-s1 and ssrna 10-s2 (see Material and methods section), show similar adsorbed amounts to the supported lipid bilayer, and we conclude that the ssrna 10 adsorption does not show any measurable dependence on the nucleic acid sequence. Another important observation is that there is no detectable adsorption of the nucleic acids to the clean bare SiO 2 surface (data not shown), which also agrees with previous reports. 39 This facilitates the quantitative interpretations of the data for DNA and RNA adsorption to the deposited bilayers. In Fig. 3, we see that for both ssrna 10-polyA and dsdna 2000, the adsorbed amount increases with increasing bulk concentration at the lower nucleic acid bulk concentrations (<0.05 mg ml 1 ). At higher nucleic acid concentrations, a plateau value is reached, and a further increase of the nucleic acid bulk concentration does not lead to further adsorption of ssrna 10-polyA or dsdna 2000 to the bilayer. Even at very high concentrations of dsdna 2000 in the solution (0.5 mg ml 1 ), the adsorbed amount is not changed. We can thus conclude that we reach a saturation adsorption of the nucleic acids to mixed bilayers at bulk concentrations #0.05 mg ml 1. The plateau value of adsorption was estimated to be ng cm 2 for ssrna 10-polyA and ng cm 2 for dsdna 2000, respectively. For all further QCM-D experiments the nucleic acid concentration used is 0.1 mg ml 1, which is well above the lowest concentration needed to reach maximum (plateau) adsorption to the deposited bilayer. In all experiments described in Fig. 3, slight nucleic acid desorption was observed when replacing the nucleic acid solution with neat buffer (rinsing). Fig. 3 Adsorption of ssrna 10-polyA and dsdna 2000 to mixed DOPC DOTAP 30 : 70 bilayers for varying bulk concentrations of RNA or DNA. The bilayer is saturated with DNA or RNA at a nucleic acid concentration of 0.05 mg ml 1. T ¼ 25 C Soft Matter, 2012, 8, This journal is ª The Royal Society of Chemistry 2012

6 Adsorption of ssrna and dsdna to mixed lipid bilayers: effect of cationic lipids The amount of cationic lipids in the deposited bilayer affects the adsorption of oppositely charged nucleic acids. We studied the adsorption of ssrna 10-polyA and dsdna 2000 to mixed bilayers with varying amounts of cationic lipids surfactants. For these studies, we used two different types of cationic components, DOTAP and sphingosine, which are quite different with respect to their chemical and physical properties. DOTAP is a synthetic surfactant with two unsaturated acyl-chains and a low melting temperature (T m ¼ 12 C), and sphingosine is a lipid with one single saturated acyl-chain and a high melting temperature (T m ¼ 36 C). As shown in Fig. 1, the mixtures of these cationic amphiphiles with a zwitterionic phospholipid with two unsaturated acyl-chains, DOPC, are very similar with respect to the area per acyl-chain in the supported bilayers. DSC experiments further show that the melting transition is below 25 C for mixtures of DOPC DOTAP vesicles and DOPC sphingosine vesicles containing up to 50% sphingosine (Table 1). The results for RNA and DNA adsorption to the lipid bilayers with different compositions are summarized in Fig. 4 and S2 in the ESI. Fig. 4a shows how the ratio between positive charges from the supported bilayer and negative charges in the adsorbed layer, /+, varies with bilayer charge density for the DOPC DOTAP and the DOPC sphingosine mixed bilayers. The corresponding data for the adsorption are represented as the number of DNA or RNA nucleotides (or bases) per lipid in the bilayer versus the percentage of lipid acyl-chains that are associated with one charged headgroup as shown in Fig. 4b. We first note that there is no significant difference between the adsorption to the mixed lipid bilayers containing DOTAP and the ones containing sphingosine. From Fig. 4a, we further conclude that there is a clear difference between the adsorption of the small ssrna 10-polyA and the large dsdna 2000 at low bilayer charges. For the larger DNA molecule, the ratio of /+ charges is high at bilayers where less than 20 25% of the lipid acyl-chains are associated with one headgroup charge. However, for the significantly smaller RNA molecules, the ratio of /+ charges is roughly the same for all bilayers containing more than 10% charged lipids (counted per single chain). Below a charge density where 10% of the acyl-chains are associated with charged groups, the amount of adsorbed ssrna 10-polyA is lower. Fig. 4 also shows that the bilayer surface appears saturated with nucleic acid when the bilayer contains more than 20% charges in terms of acyl-chain from charged lipids. A further increase in the amount of charged lipids in the bilayer does not lead to further adsorption of nucleic acids. We finally note that the adsorbed amount of dsdna 2000 is slightly higher compared to ssrna 10-polyA. Desorption of ssrna 10-polyA and of dsdna 2000 with up to 14 3% of the adsorbed material upon rinsing with buffer was observed for all mixed bilayers studied. However, the nucleic acid desorption decreases with increasing charge density of the bilayer. For all further QCM-D experiments, the bilayer composition was chosen so that 30% of the lipid acyl-chains are associated with a charged headgroup. This is well above the saturation limit in Fig. 4. The adsorption of ssrna to bilayers containing sphingosine was also visualized with fluorescence confocal microscopy using Fig. 4 Adsorption of ssrna 10-polyA and of dsdna 2000 to DOPC DOTAP and DOPC sphingosine bilayers with varying amounts of charged lipids. Data are presented both as (a) the number of nucleic acid negative charges per lipid surfactant positive charge in the outer bilayer ( /+), and as (b) the amount of nucleotides per lipid. The percentage of charged chains refers to the number of headgroup charges averaged over the number of chains. The bilayer is saturated with nucleic acids when the % charged chains exceed 20%. The concentration of RNA or DNA in bulk solution was 0.1 mg ml 1. T ¼ 25 C. giant unilamellar vesicles (GUV s). Fig. 5 shows a GUV vesicle composed of DOPC sphingosine (80 : 20) in the presence of ssrna 10-polyA. The vesicles are visualized by adding 1 mol% of a fluorescent lipid analogue, Rh-PE (red), to the lipid mixture, and Fig. 5 Confocal microscopy image of DOCP sphingosine 80 : 20 giant vesicles in the presence of ssrna 10-polyA. The bilayer contains 1 mol% fluorescent lipid analogue, Rh-PE (red), and the RNA is stained with GelStarÒ (green) after association. (a) Green channel. (b) Red channel. (c) Overlay of green and red channels, showing co-localization. The experiment shows that the RNA is accumulated at the bilayer and that the vesicles remain intact after RNA association. Scale bar: 20 mm. T ¼ 25 C. This journal is ª The Royal Society of Chemistry 2012 Soft Matter, 2012, 8,

7 the RNA is stained using GelStarÒ (green) nucleic acid dye. The images show that the ssrna is present at the bilayer, and that the presence of ssrna does not appear to induce membrane rupture during the time of the experiments. This is also consistent with only very minor changes in the dissipation in the QCM-D experiments. Adsorption of ssrna and dsdna to mixed lipid bilayers: effect of lipid acyl-chain packing In the next set of experiments, we studied the adsorption of ssrna 10-polyA and dsdna 2000 to mixed lipid bilayers with different phase behaviour. Temperature during the measurements was set to 25 C. Four different lipid systems were investigated, the zwitterionic component is either DOPC (unsaturated chains, T m ¼ 20 C) or DPPC (saturated chains, T m ¼ 41 C) and the cationic component is sphingosine (T m ¼ 36 C) or DOTAP (T m ¼ 12 C). The bulk phase behaviour of the lipid mixtures at these compositions was studied by means of DSC to determine the solid fluid phase transition temperature, and the data are presented in Table 1 and Fig. S3 in the ESI. The lipid bilayers composed of DOPC Sphingosine and DPPC DOTAP all form fluid bilayers at room temperature, while the DPPC sphingosine mixture forms a solid lamellar phase. We also note that sphingosine shows nonideal mixing with both PC lipids, as implied from the broad two-phase regions observed in the DSC measurements. From the QCM data for the deposited bilayers shown in Table 1, we conclude that the area per acyl-chain is similar for the mixed fluid bilayers with an average area of approximately A 2 per chain (note that the area per double chained lipid is twice this value), while the solid DPPC sphingosine mixed bilayer has a smaller area per chain of around 20 A 2 per chain, due to the closer acyl-chain packing in the solid phase. The measured area per hydrocarbon chain is in good agreement with previously reported data for the area per acyl-chain in different lipid phases. 43,44 Table 1 also shows data for ssrna adsorption to bilayers composed of 100% cationic lipids surfactants that either form fluid phase bilayers (100% DOTAP) or solid phase bilayers (mixture of DODAB sphingosine 85/15). DODAB is a cationic surfactant with two saturated acyl chains and with a T m of 39 C (as determined from DSC experiments). For the cationic DOTAP fluid phase bilayer, the area per acyl chain is large (35 A 2 per chain), which is slightly larger compared to the fluid phase bilayers formed in mixtures with DOPC or DPPC (around A 2 per chain). For the cationic DODAB sphingosine solid phase bilayer, the area per acyl chain is 33 A 2 per chain, a considerably larger area per chain than what has previously been observed for deposited bilayers of zwitterionic DPPC (24 A 2 per chain). 43 The higher area per chain shows a lower surface coverage of the solid phase bilayer composed of solely cationic lipids compared to the deposited bilayers with lower charge density. This can be explained by stronger repulsion between the headgroups in the bilayer, resulting in less efficient packing. Furthermore, the highly charged vesicles will experience a large lateral electrostatic repulsion as they attach to the surface, before spreading and formation of the supported bilayer. This may give rise to bare patches of SiO 2 surface. It is important here to point out that RNA and DNA do not adsorb to the bare SiO 2 surface, and it is therefore possible to make quantitative comparisons between the results obtained for bilayers with different lipid compositions if data are presented as the adsorbed amount of nucleic acid counted per deposited lipid. The adsorption of ssrna (counted as the number of bases per lipid or /+ ratio) is higher for the highly charged bilayers compared to the bilayers with lower charge density. This is observed for both solid and fluid supported bilayers. Another important observation is that there is no significant desorption of ssrna from the fully charged bilayer surface upon rinsing with buffer. Base-paring of complementary ssrna strands in bulk In the studies of RNA base-pairing, we use two complementary strands of ssrna 10, i.e. ssrna 10-s1 with sequence UCUU CUACUU and the complementary ssrna 10-s2 with sequence AAGUAGAAGA. The 10 bp dsrna 10 duplex formed has a denaturation temperature of 26 C, and the temperature for the base pairing experiments was therefore set to 20 C. Before studying the base-pairing reaction at the bilayer surface, we confirmed that this reaction occurs readily and in a stoichiometric way in bulk solution. Here, we used isothermal titration calorimetry (ITC) to monitor the base pairing reaction in buffer solution. Fig. 6 shows the enthalpy trace for the strand association by injection of ssrna 10-s1 into a solution that contains ssrna 10-s2. The measured enthalpy arises from the association of the two complementary strands. The ITC data show that the assembly process occurs within minutes after the injection, which is also in good agreement with previous studies on base-pairing of short oligonucleotides. 22,45 The upper part of Fig. 6 shows the heat detected per injection and in the lower panel both integrated and fitted data from the ITC traces are shown. From these data, the value for DH and the stoichiometry of the duplex formation was calculated to be DH ¼ 65 kj mol 1 and 1.1 stoichiometry, respectively (average from 2 measurements). Base-paring of complementary ssrna strands at deposited lipid bilayers Finally, we studied the base-pairing reaction of two complementary ssrna 10 strands that takes place at a bilayer surface. In these studies, we compared lipid systems that form either fluid or solid phase bilayers. The experiments are performed under conditions where the bilayer surface is saturated with ssrna, and where the ssrna desorption upon rinsing with buffer is negligible. If ssrna is washed away from the bilayer, or if the bilayer surface is not saturated with ssrna, it is difficult to distinguish between the base pairing reaction and non-specific adsorption of RNA directly to the bilayer. Based on the results in Fig. 3 and 4, we find conditions where the concentration of ssrna 10 in the solution and the contents of charged lipids in the bilayer are high enough to assure that the bilayer surface is saturated with ssrna 10. From Table 1 we further conclude that the ssrna 10 adsorbs to both fluid and solid phase bilayers. Finally, we find that the condition of negligible desorption of ssrna 10 is only fulfilled for highly charged bilayers. Based on these observations we chose the bilayer systems composed of 100% cationic lipids surfactants for the base-pairing studies. Comparison experiments were also performed with mixed Soft Matter, 2012, 8, This journal is ª The Royal Society of Chemistry 2012

8 the complementary ssrna 10-s2 strand as was also detected in bulk by means of ITC (Fig. 6). We note that the QCM-D cell is thoroughly rinsed with buffer between subsequent additions of ssrna to assure that there is no ssrna 10-s1 remaining in the bulk to base pair with the complementary ssrna 10-s2 strand. Fig. 6 ITC titration trace for duplex formation. (a) Raw thermogram, each peak corresponds to the injection of 10 ml ssrna 10-s1 solution into the ssrna 10-s2 solution in the calorimetric cell, except for the first peak in which only 2 ml was injected. The process is fast and the signal rapidly goes back to the baseline level after injection. This indicates that the base pairing of such a short RNA strand is fast. (b) Integrated injection data (solid squares) and a least-squares fit of the data to a one-site binding. The association enthalpy for the formation of the duplex was determined to be 65 kj mol 1 at 20 C, and the stoichiometry is determined by fitting to be 1.1 (average from 2 measurements). T ¼ 25 C. bilayers composed of DOPC sphingosine 50 : 50 and DPPC sphingosine 50 : 50. Fig. 7a shows QCM data for the experiments where ssrna 10-s1 and ssrna 10-s2 were added sequentially to the deposited cationic DODAB sphingosine (85 : 15) solid phase bilayer. The first arrow (i) indicates the addition of ssrna 10-s1 to the bulk solution. This addition leads to an increase in adsorbed mass with 88 ng cm 2. No significant desorption of ssrna 10- s1 is observed upon rinsing with buffer (ii). ssrna 10-s1 is then added again (iii) and thereafter the surface is rinsed in buffer again (iv). No significant change in the adsorbed amount is detected for any of these steps. Finally, the complementary ssrna 10-s2 is added, and there is again a significant increase (84 ng cm 2 ) of the adsorbed mass (v). It is noted that the adsorbed amount is almost doubled after the addition of ssrna 10-s2, and the data in Fig. 7a can be interpreted as the base-pairing reaction between the adsorbed ssrna 10-s1 and Fig. 7 QCM traces of the base pairing of the complementary ss strands of RNA at (a) a cationic DODAB sphingosine (85 : 15) deposited solid bilayer and (b) a cationic DOTAP deposited L a bilayer: (i) addition of ssrna 10-s1. (ii) Rinsing with buffer solution. (iii) Addition of the same RNA strand, ssrna 10-s1. (iv) Rinsing with buffer solution. (v) Addition of the complementary RNA strand, ssrna 10-s2. (c) QCM traces of the base pairing of the complementary ss strands of RNA at a deposited layer of cationic PAMAM dendrimers: (i) addition of ssrna 10-s1. (ii) Rinsing with buffer solution. (iii) Addition of the complementary RNA strand, ssrna 10-s2. The adsorbed amount of nucleic acid was calculated from the 7 th overtone using the Sauerbrey expression. T ¼ 20 C. This journal is ª The Royal Society of Chemistry 2012 Soft Matter, 2012, 8,

9 The situation is clearly different when the bilayer is present in a fluid phase following the same experiment procedure as described above. Fig. 7b shows the corresponding experimental data for the base-paring reaction on a cationic DOTAP fluid L a bilayer. The addition of ssrna 10-s1 (i) leads to an increase in adsorbed mass with 91 ng cm 2. Again, there is no significant desorption detected upon rinsing with buffer (ii). The addition of the same ssrna 10-s1 strand (iii) does not lead to any further increase in adsorbed mass. After another rinsing (iv), the complementary ssrna 10-s2 strand was added (v). Only a very small increase in the adsorbed amount could be detected. It is noted that there is a slow increase in adsorbed mass over time, and after 10 minutes the adsorbed mass has increased by ca. 20 ng cm 2. This indicates that the complementary ssrna 10-s2 strand is not able to base pair quantitatively with the already adsorbed ssrna 10-s1. We therefore conclude that we are not able to efficiently perform the base-pairing reaction on the highly charged DOTAP fluid bilayer. Similar results were also obtained on a soft cationic polymer layer of deposited PAMAM G4 dendrimers (Fig. 7c). The adsorbed layer of PAMAM dendrimers on the SiO 2 surface has previously been characterized. 46 These reported ellipsometry and neutron reflectometry data show that the G4 dendrimers deform substantially upon adsorption and form a layer with rather high surface coverage. This suggests that the dendrimers are soft and likely expose parts of the more hydrophobic ethylenediamine core upon adsorption. The report also shows that DNA readily adsorbs to this layer and forms a rather thin and compact DNA layer. These results are also interesting from another point of view, namely as cationic dendrimers have been shown to be promising synthetic vectors in gene therapy (cf. ref. 46). The experiments in Fig. 7a and b were reproduced as separate measurements where either ssrna 10-s2 or ssrna 10-s1 was added to the bilayer surface saturated with ssrna 10-s1 and not done in two subsequent steps as in the experiments shown in Fig. 7. The control experiments yielded the same results as shown in Fig. 7b with no increase in the adsorbed amount after addition of ssrna 10-s1 and almost double the adsorbed amount after the addition of ssrna 10-s2. Base-pairing studies were also performed on mixed DPPC sphingosine (solid) and DOPC sphingosine (liquid) 50 : 50 bilayers where the charge density is lower. The same trend is observed for the mixed zwitterionic cationic bilayers but the results are less unambiguous as one also has to consider the effect of desorption of the adsorbed ssrna 10 upon rinsing. Discussion The major conclusion from this study is that it is possible to control ssrna base-pairing on a charged lipid bilayer scaffold by simply changing the lipid phase behaviour. We have found conditions where the bilayer surface can be used as a scaffold for the quantitative base-pairing between complementary short ssrna 10-s1 and ssrna 10-s2 strands. The optimal conditions for base-pairing in the investigated system are found for solid phase bilayers composed of only cationic amphiphiles. When the cationic bilayer forms a fluid phase, only minor association of the complementary strands could be observed. Even less adsorption of the complementary strand was detected when the first strand was pre-adsorbed to a soft polymer layer of deposited cationic dendrimers. In previous studies, we have demonstrated that ssrna 10 penetrates into expanded lipid monolayers with exposed acyl-chains, while for condensed monolayers there is no penetration and ssrna 10 adsorption only occurs in the layer adjacent to the lipid headgroup. 20 The findings for lipid monolayers can be translated to bilayer systems, as there can be significant exposure of hydrophobic acyl-chains also in liquid crystalline bilayers. 47 One explanation for the present results on RNA base pairing is therefore that localization and penetration of ssrna 10 are different in solid and fluid bilayers. When ssrna 10-s1 is adsorbed to the bilayer with fluid acyl-chains, the ssrna bases, which are (partly) hydrophobic, can penetrate into the more hydrophobic regions in the bilayer interface, and thereby cannot pair up with the bases in the complementary strand. A similar arrangement can be expected for ssrna adsorbed to the soft layer of dendrimers with ethylenediamine core. At the solid bilayer, on the other hand, ssrna 10 cannot penetrate into the layer, which was also confirmed by neutron reflectometry in the monolayer model. 48 We therefore expect that the bases are more accessible for the complementary strand. It is also important to note that the dynamics is very different in the fluid and solid bilayers, and that the observed effects might also be (partly) related to slower kinetics of the base pairing reaction due to the high lateral diffusion in the plan of the fluid bilayer. 49 This might explain the minor gradual increase in the adsorbed amount detected after addition of the complementary strand for the fluid bilayers [(v) in Fig. 7b]. Base pairing/hybridization of DNA oligomers at surfaces has previously been reported The major difference between these studies and our experiment is that one of the DNA strands has been chemically grafted to the surface prior to base pairing with the complementary strand. DNA hybridization at bilayers by coupling a DNA strand to a biotin modified bilayer has also been reported. 36 One of the advantages of using supported bilayers as a scaffold for the base pairing reaction is that it is possible to change the surface properties and thereby control RNA association and propensity for base pairing reaction. In this way, the alteration of bilayer properties can be used as an on/off switch for the reaction. The design of the base-pairing experiments relies on careful characterization of the adsorption of nucleic acids to deposited lipid bilayers. We explored adsorption of ssrna 10 and dsdna 2000 to liquid and solid phase bilayer with different lipid composition and charge density to find conditions where the adsorption of nucleic acid at the bilayer/aqueous interface is maximal, and when desorption of nucleic acids upon replacing the nucleic acid solution with neat buffer is minimal. It is of utmost importance for the interpretation of the base-pairing experiments to find these conditions as it otherwise will be impossible to distinguish between base-pairing of a complementary ssrna 10-s2 strand to a pre-adsorbed ssrna 10-s2 strand, and adsorption of just another ssrna 10 to the oppositely charged bilayer. While deposition of cationic DODAB (solid phase) to SiO 2 surfaces has previously been reported by Pereira et al., 53 we were able to deposit both solid and fluid phase bilayers composed of solely cationic lipids on the SiO 2 coated QCM-D crystals. The adsorption of the negatively charged nucleic acids to the positively charged lipid bilayer is dominated by electrostatic Soft Matter, 2012, 8, This journal is ª The Royal Society of Chemistry 2012

10 attraction, and the adsorbed amount is dependent on the nucleic acid bulk concentration and the charge density of the deposited bilayer. As shown in Fig. 4b, the bilayer surface appears saturated with nucleic acids when the amount of lipid acyl-chains that are associated with charged headgroups is more than 10%, and a further increase in bilayer charge density does not lead to further adsorption. This is true both for sphingosine and DOTAP containing bilayers and similar trends are observed for both dsdna 2000 and ssrna 10. When the data in Fig. 4b are presented instead as the nucleic acid negative charges per lipid positive charges in the adsorbed layer (Fig. 4a), we see that we end up with an electroneutral layer (charge ratio close to 1) for most situations investigated. No significant change in dissipation was detected for any of the systems presented here, which implies that the RNA and DNA adsorb to the bilayer in a flat manner. One clear exception to this is the adsorption of dsdna 2000 to the bilayer with low charge density (less than 10% of the chains belongs to charged lipids), which leads to significant charge reversal of the bilayer, giving a highly negative net-charge. This can likely be explained on the basis of the size of the dsdna 2000 molecule. When a single dsdna 2000 molecule adsorbs to the bilayer, it brings at once 4000 negative charges to the surface, which leads to a high /+ ratio when the bilayer charge density is low. For the highly charged bilayers, the dsdna 2000 was adsorbed as a thin layer parallel to the lipid bilayer aqueous interface, in a similar way as previously proposed for DNA adsorption to zwitterionic bilayers in the presence and absence of divalent cations. 39 The result is that the DNA charges match those of the surfactant bilayer, which results in a zero net-charge density. We also conclude that the adsorbed amount of dsdna 2000 is higher than the adsorbed amount of ssrna 10-polyA for all comparable situations, which is likely be explained by the higher charge density of the double-stranded long DNA compared to the single-stranded nucleic acid species. 54 The electrostatic attractive force that drives the adsorption of the nucleic acids to the oppositely charged bilayer is thus expected to be weaker for the ssrna 10-polyA than for the dsdna The summary of the data presented in Fig. 3 and 4 shows that the adsorption of ssrna 10-polyA and dsdna 2000 can be controlled by variations in the bilayer charge density and the nucleic acid concentration within some concentration regimes. However, when the amount of charged lipids or nucleic acid has reached a certain level, the adsorption becomes virtually independent of the content of charged lipid or the concentration of nucleic acid. The balance of electrostatic attraction between the surface and the nucleic acids and the lateral repulsive forces (e.g., electrostatic repulsion between adsorbed nucleic acids and entropic steric repulsion) is the likely explanation for the saturation in adsorption, that is, the plateau value observed in Fig. 3 and 4. It is possible that the DNA strands are arranged in an ordered manner at the bilayer surface, similar to what has previously been reported for dsdna at cationic monolayers. 55 It has also been shown that dsdna strands arrange as a 2-D smectic phase in a 3- D DOTAP DNA complex, 56 while short RNA molecules, like the sirna, show no 2-D ordering when complexed with DOTAP. 57 The effect of lipid phase behaviour on the adsorption of nucleic acids to the deposited bilayers was also investigated. We find that both ssrna 10 and dsdna 2000 show slightly higher adsorption to the fluid bilayers compared to the solid bilayer. In lipid bilayers with the acyl-chains in a fluid state, the bilayer interface is dynamic and there is clear water hydrocarbon contact even for a bilayer with a high surface coverage. This is inherent in the system and has been demonstrated in molecular dynamics and coarse grain simulations as well as neutron diffraction studies. 47,58,59 In a solid bilayer, on the other hand, the acyl-chains are frozen and the lipids are more densely packed, and the hydrophobic interior is thus less exposed. It is clear that, for a bilayer with a given fraction of charged lipids, the solid phase bilayer has a higher surface charge density compared to the fluid bilayer, as the area per lipid is smaller. Still we do not expect larger adsorption to the solid bilayer with the highest surface charge density, as all systems investigated in Table 1 contain a much large fraction of cationic amphiphiles required to obtain saturation in nucleic acid adsorption. It is possible that the observed small difference in nucleic adsorption presented in Table 1 can be explained by stronger hydrophobic attraction to the fluid bilayer compared to the solid bilayer. This contribution is expected to be most important for the adsorption of ssrna with exposed hydrophobic bases. The differences observed for fluid and solid bilayers composed of 100% cationic lipids can be explained by the fact that bilayer coverage is lower for the fluid bilayer systems. Conclusions RNA lipid interactions are central to the understanding of structure and function in biological systems and to the development of new applications in medicine and biotechnology. In the present study we study adsorption and base pairing of short RNA oligonucleotides to model lipid membranes with different composition. We use model systems of supported lipid bilayers and unilamellar vesicles, and we use a combination of experimental techniques, including QCM-D, confocal microscopy and ITC. Our main conclusions are as follows: (i) Base pairing of the complementary short ssrna occurs readily in bulk when the temperature is below the melting temperature of the nucleic acid duplex (followed by ITC). (ii) Base pairing of short complementary RNA strands can be controlled by the acyl-chain packing in a deposited bilayer. It was shown that the base pairing with complementary strands takes place at the bilayer when the first strand is pre-adsorbed to a bilayer with solid chains, but not when the first strand is preadsorbed to a liquid crystalline bilayer with fluid chains. The results imply that the ssrna hydrophobic bases are not accessible to the complementary bases when RNA is associated with fluid bilayers, which can be due to hydrophobic interactions with the exposed apolar parts of the bilayer, as recently shown in lipid monolayer systems. 48 It is also likely that the difference in phase behaviour affects the kinetics for the base pairing process. (iii) The chemical nature of the different cationic amphiphiles studied here has very little effect on the adsorption behaviour of the ssrna and dsdna. Of particular interest is that we cannot observe any significant differences in the nucleic acid adsorption to bilayers that contain the naturally occurring cationic lipid sphingosine compared to bilayers that contain more conventional surfactants. (iv) The phase behaviour of the lipid bilayer influences the adsorption of nucleic acids, where there is less material adsorbed to solid bilayers compared to fluid bilayers, as measured by the relation of nucleic acid base per lipid. At saturation, the surface is This journal is ª The Royal Society of Chemistry 2012 Soft Matter, 2012, 8,