RNA and DNA Association to Zwitterionic and Charged Monolayers at

Article pubs.acs.org/Langmuir

RNA and DNA Association to Zwitterionic and Charged Monolayers at the Air−Liquid Interface Agnes Michanek,*,† Marianna Yanez,† Hanna Wacklin,§ Arwel Hughes,‡ Tommy Nylander,† and Emma Sparr† †

Division of Physical Chemistry, Center of Chemistry and Chemical Engineering, Lund University, P.O. Box 124, 22100 Lund, Sweden ‡ European Spallation Source ESS, AB - Box 176, 22100 Lund, Sweden § Rutherford Appleton Lab, ISIS Facil, Didcot OX11 0QX, Oxon, England S Supporting Information *

ABSTRACT: The objective of this work is to establish under which conditions short RNA molecules (similar to miRNA) associate with zwitterionic phospholipids and how this differs from the association with cationic surfactants. We study how the base pairing (i.e., single stranded versus double stranded nucleic acids) and the length of the nucleic acid and the charge of the lipid/surfactant monolayer affect the association behavior. For this purpose, we study the adsorption of nucleic acids to monolayers composed of dipalmitoyl phosphatidylcholine (DPPC) or dioctadecyl-dimethyl-ammoniumbromide (DODAB) using the surface film balance, neutron reflectometry, and fluorescence microscopy. The monolayer studies with the surface film balance suggested that short single-stranded ssRNA associates with liquid expanded zwitterionic phospholipid monolayers, whereas less or no association is detected for double-stranded dsRNA and dsDNA. In order to quantify the interaction and to determine the location of the nucleic acid in the lipid/surfactant monolayer we performed neutron reflectometry measurements. It was shown that ssRNA adsorbs to and penetrates the liquid expanded monolayers, whereas there is no penetration of nucleic acids into the liquid condensed monolayer. No adsorption was detected for dsDNA to zwitterionic monolayers. On the basis of these results, we propose that the association is driven by the hydrophobic interactions between the exposed hydrophobic bases of the ssRNA and the hydrocarbon chains of the phospholipids. The addition of ssRNA also influences domain formation in the DPPC monolayer, leading to fractal-like interconnected domains. The experimental results are discussed in terms of the implication for biological processes and new leads for applications in medicine and biotechnology.

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membranes using phospholipid liposomes as vehicles.12 Recent studies have also shown that liposome-based delivery systems are effective in transporting siRNA (another short RNA) to certain types of tissue.4 To rationally approach the design of phospholipid vehicles as delivery tools for miRNA and for siRNA, one requires fundamental understanding of the interactions between phospholipids and these small RNA molecules. The characterization of RNA interaction with lipid membranes is also essential for understanding the organization and function of these molecules in living systems. During the past few years it has been revealed that the chromatin complex includes phospholipids, and that RNA indeed colocalizes with these lipids.13−15 The presence of substantial amounts of

INTRODUCTION It has in recent years become clear that short DNAs and RNAs play an important role in the regulation of for example protein expression in cells. Short RNAs are believed to be able to help in both up and down regulation of transcription and translation, for example by interfering with mRNA.1−4 One example of this is the short miRNA (or micro RNAs, that is, noncoding short RNA sequences containing 20−30 bases), which is believed to take part in several cellular processes such as cell death and cell proliferation. It has furthermore been suggested that miRNA may have an important role in the initiation of cancer.5,6 Therefore, the interest in using miRNA therapy for treatment of diseases has grown in recent years,4 which for example has led to the development of promising new anticancer miRNA drugs.7 There are several ways of delivering miRNA to the target inside the cell.8−11 One way is to use phospholipid-based drug delivery systems. Already in 1991, Akthar et al. showed that oligonucleotides can be transported across biological © 2012 American Chemical Society

Received: November 10, 2011 Revised: May 7, 2012 Published: May 24, 2012 9621

dx.doi.org/10.1021/la204431q | Langmuir 2012, 28, 9621−9633

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of interaction by varying nucleic acid length, base pairing and the monolayer charge density. Based on these results, neutron reflectometry (NR) measurements were carried out to quantify the association, and to determine the location of the nucleic acid relative to the lipid/surfactant monolayers. Fluorescence microcopy was used to study the lateral organization (domain formation) in the monolayers in the absence and presence of the nucleic acids, e.g., ssRNA. Our major objectives are to (1) Establish the role of base pairing in the interaction between nucleic acids and zwitterionic model membranes. (2) Reveal the differences in nucleic acid association to zwitterionic model membranes compared to cationic model membranes (3) Determine how the association is influenced by monolayer phase behavior and surface pressure (4) Determine how domain formation in the monolayer is influenced by the addition of nucleic acids

phospholipids in isolated cell nuclei has also been demonstrated with mass-spectroscopy studies.16 The endonuclear lipids have been related to structural function, signal transduction and in stimulating RNA or DNA synthesis,17−21 although their precise function is still not fully understood. It is most likely that endonuclear lipids interact with RNA and DNA and thus influence cellular processes. Association of RNA and DNA to biological membranes has been demonstrated for model membranes composed of zwitterionic phospholipid and cationic sphingosine22 and for zwitterionic phospholipid (PC) bilayers in the presence of divalent cations.23,24 In the latter situation, the divalent ions are supposed to associate with the PC bilayer, making the zwitterionic bilayer behave as being cationic.25,26 It has also been reported that the permeability of phospholipid membranes increases after association with RNA.23 The association has mainly been attributed to electrostatic attractions between anionic DNA (or RNA) and (apparently) cationic membranes.27,25,28,29 Only a few studies have been dedicated to the interactions of DNA and RNA with lipid systems that do not contain cationic lipids or divalent cations, as this generally implies very weak interactions.30 In our previous work we have studied the interaction of nucleic acids with zwitterionic and anionic phospholipids in the absence of divalent salt by means of differential scanning calorimetry (DSC) and dissipative quartz crystal microbalance (QCM-D). The study showed that the length of the nucleic acids is one of the crucial factors in determining how association influences the lipid phase behavior, and the largest effects were observed for the shorter nucleic acids.31 The aim of the present study is to reveal under which conditions RNA associates with zwitterionic lipid model systems relative to the association with the corresponding cationic monolayer. We study the difference between short single stranded and double stranded RNA oligonucleotides, as well as one longer dsDNA. In the comparison between the different nucleic acids, it is important to note that RNA naturally occurs in the single-stranded form more often than DNA, where the apolar parts of the bases are available to hydrophobic interactions with other species. This can explain previous studies showing that the adsorption of DNA to hydrophobic surfaces is significantly higher for single stranded and short DNA molecules compared to longer and/or double stranded DNA molecules.32 A central question in the present study is whether hydrophobic interactions also lead to the association of single-stranded nucleic acids to zwitterionic phospholipid systems. We have chosen the lipid model system to be a well-characterized phospholipid and surfactant monolayer where we can study the effect of variations in certain physical parameters (i.e., surface pressure, Π, or area per lipid molecule) in a systematic way. The nucleic acids used in this study are 10 bases long single stranded (ss) or double stranded (ds) RNA oligonucleotides with different RNA sequences (the ssRNA has a polyA or a polyU sequences, thus making it impossible for the strands to basepair, the dsRNA is specially designed not to self-associate and has the sequence UCUUCUACUU, 5′→3′). As reference systems, we also study a longer double-stranded 2000 bp DNA. The monolayers are composed either of zwitterionic dipalmitoyl phosphatidylcholine (DPPC) or cationic dioctadecyl-dimethylammoniumbromide (DODAB). The association between the nucleic acids with the lipid monolayer surface was followed using the surface film balance in order to determine the mode

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MATERIALS AND METHODS

Materials. DODAB (dioctadecyl-dimethyl-ammoniumbromide), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rh-PE), and 1,2-dipalmitoyl(D62)-sn-glycero-3-phosphocholine (d-DPPC) with 99% purity were purchased from Avanti polar lipids Inc. (Alabaster, U.S.A.). Hydrogenated 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (h-DPPC), 99% purity, was purchased from Larodan fine chemicals (Malmö, Sweden). Dimethyl-di (octadecyl-d37) ammonium bromide was purchased from CDN Isotopes (Quebec, Canada). Double stranded salmon sperm DNA (dsDNA2000, 2000 ± 500 bp) was purchased from Invitrogen Life Technologies (California, U.S.A.). Short ssRNA (ssRNA10, poly A 10 bases), short ssRNA (polyU 10 bases), and short dsRNA (dsRNA 10 , 5′3′ UCUUCUACUU, 10bp) were synthesized and HPLC purified by MWG Biotech (Ebersberg, Germany). The ssRNA sequences were considered to be extended in the solution and thus not folding back on themselves as the persistence length for single stranded oligonucleotides has been reported to be around 3 nm.33 The dsRNA10 was assembled from two complementary ssRNA strands, one with the sequence 5′3′ UCUUCUACUU and the other strand with sequence 5′3′ AAGUAGAAGA, by mixing these two ssRNA at slightly elevated temperatures (50 °C) and left to cool to room temperature. To confirm base-pairing, isothermal titration calorimetry (ITC) measurements (not shown) of the association enthalpy resulting from the association of the two complementary RNA strands (sequence described above) were performed. The results showed that the assembly process is fast, i.e., it occurs within minutes, which is in good agreement with previous studies of the base-pairing of short oligonucleotides.34,35 The ssRNA sequence was designed specifically to avoid self-association and mismatching, whereas the complementary strands will assemble without mismatching upon mixing (design kindly provided by Prof. Luc Jaeger, UCSB). Imidazole (purity 99.5% or higher) was purchased from Sigma Aldrich (Missouri, U.S.A.). Chloroform and methanol (both 99.8% purity) were purchased from Merck (Darmstadt, Germany). All solutions were prepared using ultrapure water from a Milli-Q Ultrapure water purification system from Millipore (Massachusetts, U.S.A.), and D2O was obtained from Sigma Aldrich. The RNA samples were analyzed using inductively coupled plasma mass spectrometry (ICP MS), to determine the RNA concentration (P), the counterion (Na+) concentration, and the content of divalent ions (i.e., calcium, magnesium, iron, aluminum, and copper) of each sample. Analysis showed that the samples contained very low amounts of divalent ions (less than 0.039 mol Ca2+ /mol RNA nucleotides and even lower for the other multivalent ions) and counterions (ca. 4 mol Na+/ mol RNA nucleotides). One of the batches of ssRNA (the complementary strand with sequence 5′3′ AAGUAGAAGA) contained 9622


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an insoluble contamination, which was removed with ultracentrifugation, and the supernatant containing the RNA was collected. The RNA concentration in the supernatant was then determined by means of ICP MS. The same problem did not occur with the other RNA samples, which were used as received. All glassware was soaked in 10% aqueous hydrochloric acid for several hours and then washed 10 times with Milli-Q water. Glassware for sample preparation was heat sterilized before use to deactivate RNases, which are omnipresent and catalyze RNA degradation. The oligonucleotides were dissolved in 2 mM imidazole aqueous solution at pH 7.0 for all experiments in the present study. Langmuir Surface Film Balance. A Langmuir surface film balance (Micro through X, operated by film ware X 3.57 software, KIBRON Inc. Helsinki Finland) was used for measuring the surface pressure versus area (Π−A) isotherms as well as the surface pressure versus time (Π−time) isotherms. The through is made out of high quality sand blasted glass with a Teflon rim and has a surface area of 13 747 mm2 (233 × 59 mm) with a subphase volume between 20 and 30 mL. Two moveable Teflon barriers control the surface area and Π is recorded with the Wilhelmy method using a platinum alloy needle connected to a microbalance as surface pressure sensor. The through and the barriers were thoroughly cleaned with MQ water, followed by ethanol and then rinsed with MQ water before each measurement. In addition, the through was cleaned using the laboratory detergent Decon 90 (Decon Laboratories Limited, East Sussex, U.K.) and then rinsed with substantial amounts of MQ water in the beginning of each new set of measurements and in the beginning of each measurement day. Finally, the air/aqueous interface was aspirated clean using a suction pump. The probe used to measure surface tension was cleaned using a flame. Blank runs were performed before each measurement and the blank was considered to be good if Π remain below 0.2 mN/m at the highest compression (the minimum area). It is here noted that with the present microtrough setup, there is indeed a small change in the liquid meniscus when the barriers are compressed to the minimum area, which slightly affect the surface tension measurement at small surface areas. Therefore, the experimental conditions were adjusted so that the measurements are performed at larger surface areas. The temperature was kept at 22 °C and controlled using an external water bath. DPPC and DODAB were dissolved in chloroform/methanol, 2:1 mixture and kept as stock solutions in the freezer. Every day a fresh solution of lipid/surfactant with concentration 0.5 mg/mL was prepared using the concentrated stock solution; the stock solutions (5 mg/mL) were prepared fresh weekly. The lipid/surfactant solution (25 μL, 17 nmol DPPC, or 19.75 nmol DODAB) was spread using a Hamilton syringe and 10 min equilibration was allowed for the solvent to evaporate before starting the measurement. The compression rate was set to 5 mm/min (295 mm2/min). Two types of experiments with nucleic acids were performed. Either the nucleic acids were mixed in the subphase before the monolayer was spread, or the nucleic acids were injected into the subphase after the monolayer was spread. In the latter experiments, the monolayer was spread on the subphase containing only buffer. The monolayer was then compressed to a chosen Π and the monolayer area was then kept constant. After the injection of the nucleic acids to the subphase, the changes in Π were recorded as a function of time (at constant monolayer area). The nucleic acids were injected by carefully replacing 10 mL of the original buffer subphase with 10 mL of stock solution containing nucleic acids in the same buffer. It was found that a good procedure for doing this is to first remove 5 mL of subphase from one end of the trough outside of the barriers, then add 10 mL of the new solution on the outside of the barriers in the other end of the trough, and finally, remove another 5 mL of subphase from the same side as the first 5 mL. This protocol was found to give the most efficient and reproducible mixing of the nucleic acid stock solution with the subphase, compared with several different procedures tested. The final concentration of the different nucleic acids in the subphase was 0.06 mg/mL. The Π−time measurements were run for approximately 90 min followed by the recording of a Π−A isotherm. All experiments

shown here were reproduced at least three times with similar results, and we show data from one representative experiment. Neutron Reflectometry. Measurement of specular reflection of neutrons enables the characterization of the structure normal to an interface. Here we will describe the main features of the technique and more detailed account of the theory and applicability is given by, e.g., Thomas.36 Neutron reflectometry measures the intensity of reflected neutrons as a function of the scattering vector Q = (4π sin θ)/λ, where θ is the angle of incidence and λ is the neutron wavelength. On the basis of these data the neutron scattering length density profile perpendicular to the surface of the layer at the air/water interface can be determined. One important feature of neutron scattering is that neutrons interact with the nucleus of an atom and therefore different isotopes of the same element do scatter differently, i.e. have different scattering length density. The difference in their coherent scattering is particularly large between hydrogen and deuterium, for which the neutron scattering lengths are −3.742 × 10−2 and 6.67 × 10−5 Å, respectively. Contrast variation can be achieved by isotopic substitution, particularly by deuterium substitution for hydrogen.

Table 1. Parameters Used for Fitting the Neutron Reflectivity Data component D2O nrw h-DODAB d-DODAB h-DPPC d-DPPC ssRNA10 in H2O ssRNA10 in D2O dsDNA2000 in H2O dsDNA2000 in D2O

molecular volume (Å3)

SLD (10−6) (Å−2)

630.95 705.41 734.04 869.63 347 × 10a 347 × 10a 652 × 2000b

1106 1106 1216 1216 3150c 3150c 1 257 700d

6.35 0.00 −0.34 6.23 0.22 5.53 3.6 4.5 3.7

652 × 2000b

1 257 700d

4.1

molecular weight (g/mol)

a Calculated for ssRNA10, poly A, 10 bases. bCalculated as the mean of possible basepairs for the 2000 bp Salmon sperm DNA used in this study. cMolecular volume of ssRNA10 (poly-A) from Voss et al.62 d Molecular volume of dsRNA2000 calculated as the mean of possible basepairs using from Nadassy et al.63

Table 1 shows the scattering length densities and the molecular volumes for the solvents and compounds used in this study. The null reflecting water (nrw) has the composition of 92 mol % H2O and 8 mol % D2O, and this solvent has a scattering length density of zero, which is the same as that for air. The scattering length density (ρ) of a material is expressed as

ρ=

∑ nibi

where bi is the scattering length of component i and ni is the number of nuclei in a given volume, which for a compound i can be calculated from the molecular volume, Vm,i as ni = 1/Vm,i.36 Calculated profiles of the reflectivity versus the scattering vector Q were fitted to the experimental data using Motofit,37 which uses the Abeles optical matrix method38 to calculate the reflectivity of thin layers and enables the global fitting of data sets of different isotopic compositions. A one-layer slab model was used for the lipid/surfactant monolayers and a two-layer model was used for the monolayers in the presence of the nucleic acids. The thickness (d), the interfacial roughness (δ), the scattering length density of the material (ρ), and the coverage or, equivalently, the “solvent” volume fraction in the layer (φ) was used as the fitting parameters. The area per molecule (A) can be then be calculated as 9623


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Figure 1. (a) Π vs area compression isotherm of DODAB monolayers spread on subphase containing 2 mM imidazole buffer (pH 7.0) in the presence of nucleic acids premixed in the subphase (concentration 0.06 mg/mL) and on neat buffer. The presences of nucleic acids in the subphase only have minor effect on the isotherm shape. (b) Π vs area compression isotherm of DPPC monolayer spread on a subphase with 2 mM imidazole buffer (pH 7.0) and fluorescence microscopy images taken at different Π. At low Π the monolayer is in the gaseous state and the surface appear bright in the fluorescence images. In the coexistence region between LE and LC phase characteristic DPPC domains are visible at the surface. At higher Π within the plateau regime, the size of the LC domains increases significantly. It is not possible to image the LC phase as the dye is excluded from the domains and the image appears dark.

A=

∑ i

bi

made it possible to change the surface area of the monolayer at the same time as images of the surface was recorded. DPPC was mixed with a fluorescent phospholipid analogue, Rh-PE, at a molar ratio of 100:1. Images were simultaneously with the compression isotherms and as well as during the Π−time measurements. Changes in the domain shape/organization were analyzed after the addition of different nucleic acids. Control experiments, where pure buffer solution was added instead of the nucleic acid solution, were also performed.

ρlayer d

where ρlayer is the total scattering length of the layer. For a mixed layer of lipid and NA we can calculate the ρlayer as

ρlayer = ρlipid ϕlipid + ρNA ϕNA + ρsolv ϕsolv This can also be converted into the lipid to NA molar ratio as nlipid nNA

=

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(d1ϕNA,1 + d 2ϕNA,2)/Vm ,NA

RESULTS In this study we investigate the effect of size and base pairing on the interaction of nucleic acids with zwitterionic and cationic monolayers. The nucleic acids studied include 10 bases ssRNA’s, a 10 base-pair dsRNA, and a 2000 base-pair dsDNA. The concentration of the nucleic acids used was chosen so that the number of nucleotides (bases) and therefore the number of added anionic charges is the same in all experiments. This experimental design should facilitate quantitative comparisons so that observed differences can be directly coupled to the molecular size and basepairing rather than the number of opposite charges of the nucleic acids. Consequently, the number of ssRNA molecules is twice that of dsRNA molecules, and the number of dsDNA molecules is 1/ 200 compared to the number of ssRNA molecules. Π−Area Compression Isotherms for DODAB. Figure 1 shows Π−A isotherms for the cationic surfactant DODAB on a subphase composed of 2 mM imidazole buffer solution (pH 7.0) with different nucleic acids. When the area/molecule is above 140 Å2/molecule, DODAB forms a monolayer that exhibits a gaseous behavior. As the surface area decreases, Π starts to increase at an area of about 140 Å2/molecule. Upon further compression of the monolayer there is a change in slope of the compression isotherm at ca. 100 Å2/molecule. No “plateau region” corresponding to a first-order transition between a liquid expanded (LE) and a liquid condensed (LC) monolayer is detected under the present conditions. The presence of the different nucleic acids in the subphase only slightly affects the DOBAB compression isotherms. The isotherms obtained in the presence of dsDNA2000 and dsRNA10 indicate a slight decrease in the monolayer

(d1ϕlipid,1 + d 2ϕlipid,2)/Vm ,lipid

Neutron reflectometry measurements were performed using the SURF reflectometer at ISIS, Rutherford Appleton Laboratory, U.K., and for our experiments the instrument was equipped with 5 PTFE trough of dimensions 23.8 cm ×5.0 cm = 119 cm2. The surface pressure, Π, after spreading the lipid was checked by using a Wilhelmy plate connected to a sensor from the Nima Technologies (now KSV NIMA). The volume used was 50 mL. The measurements were made at two angles of incidence, θ = 0.5 and 1.5°, using white beam and time-of-flight (wavelength (λ) of 0.56−6.8 Å) to cover a Q range of 0.016−0.59 Å−1. A total of 32 μL of DPPC or 21 μL of DODAB solutions (0.5 mg/ mL in chloroform/MeOH 2:1) were spread at the air−liquid interface until Π reaches 8.4 ± 0.1 (for DPPC) or 8.8 ± 0.1 mN/m (for DODAB). The subphase was composed of either pure buffer or buffer solution with ssRNA10 or dsDNA2000 (0.06 mg/mL). The monolayers were left to equilibrate for 10 min in order to allow for evaporation of the spreading solvent. The reflectivity vs Q was recorded at constant monolayer area, and steady state adsorption was obtained already before recording the first reflectivity curve. After the steady state reflectivity curve was recorded, additional 18.5 μL of DPPC or 7.5 μL of DODAB solution was spread, which for the pure lipid/surfactant monolayer corresponded to a Π of 28 ± 0.1 mN/m. The reflectivity vs Q was then recorded again for the monolayer at higher Π. Experiments were performed using buffers prepared using D2O or null reflecting water (nrw, water contrast with scattering length density of 0), and both deuterated and hydrogenated lipids/surfactants were used. Fluorescence Microscopy. The domain formation in the DPPC monolayer was studied using an Axioplan microscope with a LD Achroplan 40×/0.60 Korr objective from Zeiss. The samples were illuminated using a HB100 light source, and images were collected using an Axiocam MRm digital camera operated by the Axiovision software. The Langmuir through was fitted onto the microscope which 9624


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The ΔΠ-time isotherms for the DODAB LE monolayer after the addition of dsDNA2000 or dsRNA10 can be described in terms of two regimes; first a monotonic increase in Π with time, and then an equilibration regime where Π slowly approaches a plateau value. For dsDNA2000, an increase in Π reaching a plateau value of ΔΠ = 4.0 ± 0.1 mN/m was reached after 50 min. This is very similar to the results obtained for of dsRNA10, for which the plateau value of ΔΠ = 3.8 ± 0.1 mN/m was reached after 60−70 min. In contrast to ssRNA10, we never observe a drop in Π for neither dsRNA10 nor dsDNA2000. Experiments where the nucleic acids were injected into the subphase without any lipid/surfactant monolayer were also exerted (data not shown). It was concluded that none of the nucleic acids investigated give rise to any detectible changes in the surface tension and thus their adsorption to the bare air− water interface is insignificant. ssRNA10 Adsorbs into DPPC Monolayers. The association of RNA to lipid/surfactant monolayer can be influenced both by monolayer composition and monolayer properties. Apart from the effect of lipid headgroup charge, properties like the acyl-chain packing and monolayer domain formation can also influence the association. These properties of the monolayer can be controlled in the Langmuir film balance in combination with fluorescence microscopy. As shown in Figure 1b, the zwitterionic DPPC monolayers show a rich phase behavior, which features formation of monolayer domains, as function of the area per molecule, which is also in good agreement with previously reported data on DPPC with various subphases.39−41 We therefore studied the influence of nucleic acids on monolayers of DPPC as a function of the initial Π. The experiments were performed at Πi = 6 mN/m (Figure 3a), where the monolayer forms a LE phase, at Πi = 11 mN/m (Figure 3b) where the LE and LC phases coexist, and at Πi = 17 mN/m (Figure 3c) where a LC monolayer is formed. The addition of ssRNA10 to the subphase beneath the DPPC LE monolayer results in a gradual increase in Π, and a plateau at ΔΠ = 2.1 ± 0.1 mN/m is reached after approximately 80 min (Figure 3a). Control experiments using ssRNA10 with a different sequence, polyU ssRNA10, gave similar results (not shown), thus indicating that the observed effects do not significantly depend on the RNA sequence. A small but significant increase in ΔΠ was also observed after the addition of dsRNA10, and the plateau value of ΔΠ = 0.8 ± 0.1 mN/m was reached after 90 min. The addition of dsDNA2000 did not significantly influence Π, and the results cannot be distinguished from those obtained when only buffer was injected. Similar trends were observed for the monolayer with coexisting LE and LC phases (figure 3b), where the addition of ssRNA10 leads to an increase in Π, and a plateau value of ΔΠ = 1.6 ± 0.1 mN/m reached after approximately 80 min. The injection of dsRNA10 also leads to an increase in Π, and a plateau at ΔΠ = 1.2 ± 0.1 mN/m was reached within 90 min. Again, no change in Π was detected after injection of dsDNA2000. Finally, for the DPPC LC monolayer, none of the nucleic acids investigated had any effect on Π (Figure 3c). When comparing the ΔΠ−time profiles for the LE monolayers composed of either zwitterionic DPPC (Figure 3a) or cationic DODAB (Figure 2b), we note that the addition of the different single and double stranded nucleic acids affect the monolayers in different ways. For the DODAB monolayer, the largest increase in ΔΠ was observed after the addition of the long double stranded dsDNA2000, and the smallest increase was detected after the addition of the short and single stranded

compressibility, while the isotherm recorded in the presence of ssRNA10 is almost identical to that without the nucleic acids. Adsorption of RNA and DNA to DODAB Monolayers. The influence of nucleic acids on the monolayer was studied in terms of the change in Π with time elapsed after the addition of the nucleic acid. Representative data for different nucleic acids and DODAB monolayers are shown in figure 2. Data from one

Figure 2. ΔΠ−time measurement at constant area (Πi = 9.0 ± 0.1 mN/m, aheadgroup= 97 Å2/molecule) for DODAB monolayer recorded after the injection of nucleic acids to the subphase. The increase in ΔΠ is largest for dsDNA2000 and dsRNA10. The injection of ssRNA10 deviates from the ds nucleic acids, as a dip in ΔΠ is observed immediate after injection. The symbols in the figure does not indicate individual measuring points but are merely there to clarify the positions of the curves.

control experiment (injection blank) where neat buffer was injected to the subphase instead of nucleic acid solution are also included. A small increase in Π over time due to evaporation from the subphase is observed in all blank Π-time measurements. This is taken into account in the quantitative interpretations Π versus time data. The figure shows the change in Π relative the to initial surface pressure (Πi); ΔΠ = Π − Πi. The data were obtained by first spreading DODAB on the buffer subphase and then compressing the monolayer to Πi = 9. When the monolayer had equilibrated, an aliquot of nucleic acids (corresponding to 5.45 μmol negative charges or, equivalently, 5.45 μmol nucleotide bases) was injected in the subphase. All of the nucleic acids investigated gave rise to an increase in Π, although the ΔΠ-time profiles depends on the type of nucleic acid. Interestingly, a dip in ΔΠ is observed within the first few minutes of the experiment for the single stranded ssRNA10 species but not for the double stranded nucleic acids, dsRNA10 and dsDNA2000. For the DODAB-ssRNA10 system, three different regimes are observed. Directly after the addition of ssRNA10, there is a drop in Π, which last for 5 min before reaching ΔΠ = 0. This drop in Π is reproducible, although the magnitude varies (ranging from 0.8 to 1.5 mN/m). This is followed by a regime where the Π increases linearly with time, and finally, an equilibration period in which the Π slowly approaches a plateau value as the interface becomes saturated with ssRNA10. The plateau value is reached after approximately 50 min. The final ΔΠ is 1.7 mN/m higher than the initial Π (Πi = 9.0 ± 0.1 mN/m), and the difference between the minimum (bottom of the dip) and the maximum values in Π is 2.6 ± 0.1 mN/m. 9625


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monolayer systems. This is a measure of a macroscopic thermodynamic quantity that recalls a sum of various phenomena at the interface, and this measure does not provide any molecular details. In other words, we do not obtain any information on adsorbed amount, surface structure, or penetration depth from the measures of surface tension/surface pressure. In order to confirm and quantify the adsorption of nucleic acids to the monolayers and to achieve information on the penetration of those into the monolayer, we studied selected systems by means of neutron reflectometry (NR). In order to get a good characterization of the systems, the experiments on each system investigated were performed for buffer solutions prepared of D2O and buffer solutions prepared in null reflecting water (nrw, that is 92 mol % H2O, 8 mol % D2O) and for both deuterated and hydrogenated lipids/ surfactants. The nrw solvent contrast is of particular importance for studies of layers at the air/water interface, as this solvent has a scattering length density of zero, which is the same as for air. Therefore, the reflectivity measured in this solvent contrast arises only from the adsorbed layer at the interface. Selected NR data together with the fits and the resulting scattering length density profiles for DODAB monolayer (Πi = 8.8 ± 0.2 mN/m) in the absence and presence of ssRNA10 on nrw are show in Figure 4a. Figure 4b shows the corresponding data for ssRNA10 and the DPPC monolayers (Πi = 8.4 ± 0.2 mN/m). Additional data recorded for other isotope contrasts as well as data recorded for the same systems at Πi = 28.0 ± 0.2 mN/m and for the same monolayer systems in the presence of dsDNA2000 are shown in the Supporting Information (Figure S2 and S3). A direct comparison between the NR data in Figure 4 shows that the presence of ssRNA10 clearly affects the NR profiles in the interfacial layer. The fitting parameters for the NR data are given in Tables 2 and 3. From the fitted data it is possible to calculate the scattering length density profiles, and the one for nrw is shown in the inserts in Figure 4, whereas the corresponding profiles for other isotopic contrasts are given in the Supporting Information. From these profiles it is possible to draw conclusions about the location of species with different scattering length densities respect to the interface. The results from the best fits of the NR data for all systems investigated are summarized in Tables 2 and 3, as well as in Tables S1 and S2 in the Supporting Information. The data recorded for the neat DODAB or DPPC monolayers (spread on a pure buffer solution) were fitted to a one-layer model. The best fit of the data recorded for the monolayers spread on the solution that also contain nucleic acids were obtained using a model with two layers. The strategy of the fitting is that the area per lipid/ surfactant molecule in the monolayer is determined from the data obtained for deuterated lipid/surfactant on nrw buffer. The results are then verified with another contrast, that is hydrogenated lipid/surfactant on D2O buffer. These contrasts are, however, not sufficient to get an unambiguous fit for the monolayers spread on a subphase that also contains nucleic acids. Therefore an additional contrast was used, namely deuterated lipid/surfactant on D2O. The area per molecule in the neat lipid/surfactant monolayer calculated from the data fitted to the reflectivity profiles are close to the expected area per molecule calculated from the spread amount, which verifies the evaluation strategy. The reason for the small discrepancy between calculated values and the area obtained from the fits is likely related to errors in the estimated amount spread at the air/aqueous interface. It should here be emphasized that we

Figure 3. ΔΠ vs time measurement of nucleic acid adsorption to DPPC monolayer at different initial Πi. (a) Πi = 6 mN/m (88 Å2/ molecule, LE phase). The addition of ssRNA10 leads to a significant increase in ΔΠ and the addition of dsRNA10 lead to a small increase in ΔΠ, whereas the addition of dsDNA2000 has no effect on Π that may be distinguished from the blank measurement (addition of pure buffer). (b) Πi = 11 mN/m (68 Å2/molecule, LE/LC coexistence region) injection of ssRNA10 and dsRNA10 still gives rise to an increase in ΔΠ. Upon injection of dsDNA2000, Π remain unchanged. (c) Πi = 17 mN/m (58 Å2/molecule). No increase in ΔΠ was observed for any of the injected nucleic acids.

ssRNA10. For DPPC, on the other hand, no detectable change in ΔΠ was observed after the addition of dsDNA2000, whereas the largest effect on ΔΠ was observed after the addition of ssRNA10. Quantifying the Interaction between Nucleic Acids and Lipid/Surfactant Monolayers by Neutron Reflectometry. The surface balance studies provide information of how the addition of nucleic acids affect Π for different 9626


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monitored Π during the spreading of the monolayer to make sure we arrive at the desired Π (and hence the desired area per molecule). The NR profiles reported in Figure 4a are fully consistent with the conclusion that ssRNA10 adsorbs and penetrates into the DODAB LE monolayer at Πi = 8.8 ± 0.2 mN/m. In fact, the fitting show that the monolayer contains about 35 vol % ssRNA10, if we assume that the surfactant content at the air/ liquid interface is not affected by the addition of nucleic acids. This is a reasonable assumption as the solubility of this surfactant in aqueous solution is very low. In addition to the ssRNA10 “penetrating” the monolayer, there is also a thin layer (roughly 5.5 Å) below the monolayer, most likely attached to surfactant headgroups, that contains about 58 vol % ssRNA10. This results in a total surfactant/nucleic acid molecular ratio of about 3. This result should be compared to the case where ssRNA10 is present in the subphase below the DODAB monolayer at Πi = 28.0 ± 0.1 mN/m, where the NR data imply that there is no penetration of ssRNA10 into the DODAB monolayer. However, there is still a substantial amount of ssRNA10 in the layer attached below the DODAB headgroups. The volume fraction of ssRNA10 in this layer is about 70% (Table 3 and Figure S1 in the Supporting Information). This gives a lipid/nucleic acid molecular ratio of about 13, which is close to charge neutralization. Similar behavior was observed when DPPC monolayers were spread on subphases containing ssRNA10, although the effects are considerably smaller as apparent when comparing Figure 4a,b. From the fitted data in Table 3, it is concluded that ssRNA10 adsorbs and penetrates into the DPPC LE monolayer at Πi = 8.4 ± 0.2 mN/m, whereas there is no penetration of ssRNA10 into the DPPC LC monolayer at Πi = 28.0 ± 0.2 mN/ m. The results from the fit suggest that the DPPC monolayer at low Πi contains about 20 vol % of ssRNA10 and that the volume fraction of ssRNA10 in the layer below the monolayer is about 20 vol %. This gives a total of 7.5 lipid molecules per RNA. For the DPPC LC monolayer (Πi = 28.0 ± 0.2 mN/m), on the other hand, no ssRNA10 is to be found within the monolayer, while the nucleic acid content in the layer below the DPPC headgroups is 50%. This results in a total of about 30 lipids per ssRNA10.

Figure 4. Reflectivity profiles recorded for DODAB (a) and DPPC (b) monolayers spread on buffer solution containing 2 mM imidazole, pH 7, with and without 0.06 mg/mL ssRNA10 at Πi = 8.8 and 8.4 mN/m, respectively. The scattering length density profiles corresponding to the fitted curves are inserted. The data and corresponding fit for the nrw contrast is showed. It should be noted that the fitted curves are global fits to three different contrasts and the complete data set is shown in Supporting Information.

Table 2. Properties of the Lipid Monolayers Obtained by Fitting a One Layer Model to the Neutron Reflectivity Dataa layer 1 monolayer

d1 (Å)

ρ1(10−6) (Å−2)

h-DODAB, 8.8 mN/m d-DODAB, 8.8 mN/m h-DODAB, 28 mN/m d-DODAB, 28 mN/m h-DPPC, 8.4 mN/m d-DPPC, 8.4 mN/m h-DPPC, 28 mN/m d-DPPC, 28 mN/m

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ±

20 20 20 20 22 22 22 22

2 1 2 1 2 1 2 1

0.12 3.59 0.44 4.80 0.69 3.76 0.97 5.25

0.04 0.05 0.04 0.05 0.04 0.05 0.04 0.05

ϕ1(%)

δ

ANRb (Å2/lipid)

d

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ±

5 42 ± 2 11 ± 2d 23 ± 1 9 ± 2d 32 ± 1 12 ± 2d 10 ± 1

4 4 4 4 4 4 4 4

1 1 1 1 1 1 1 1

96 96 72 72 83 83 60 60

e

3 3 2 2 3 3 2 2

Aexpc (Å2/lipid) 119 133 87 98 91 99 57 68

± ± ± ± ± ± ± ±

12 13 7 7 6 6 3 3

a The hydrogenated surfactant/lipid was recorded in D2O and the deuterated surfactant in nrw. The errors are estimates based on the significance of the fits to the neutron reflectometry data in at least three different solvent contrasts. bArea per lipid headgroup, as calculated from the fit to the reflectivity profiles. cArea per lipid headgroup calculated from the amount of lipid spread at the air−liquid interface, and the surface area. The precision of measure spreading solvent is assumed to be about ±2 μL. The evaporation of solvent before spreading the solvent is not included. dThis is the amount solvent penetrating into the monolayer in D2O contrast, where the acyl chain region is mostly assumed to be in the air with 0 scattering length density and the volume fraction of lipid is the same as for the deuterated lipid/surfactant monolayer on nrw. eThis is based on the data obtained from the measurement.

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Table 3. Properties of Lipid Monolayers Spread on a 0.06 mg/mL ss-RNA10 Obtained by Fitting a Two Layer Model to the Neutron Reflectivity Dataa layer 1

layer 2

monolayer

d1(Å)

ρ (10) (Å2)

δ (Å)

ϕLIPID,1 (%)

ϕNA,1 (%)

d2 (Å)

ρ (10) (Å2)

δ (Å)

ϕNA,2 (%)

d-DODAB, 8.8 mN/m + ssRNA10 d-DODAB, 28 mN/m + ssRNA10 d-DPPC, 8.4 mN/m + ssRNA10 d-DPPC, 28 mN/m + ssRNA10 d-DODAB, 8.8 mN/m + dsDNA2000 d-DODAB, 28 mN/m + dsDNA2000 d-DPPC, 8.4 mN/m + dsDNA2000

20 ± 1

4.86 ± 0.05

4±1

58 ± 2

35 ± 2

6±1

2.07 ± 0.05

2±1

58 ± 2

3±1

20 ± 1

4.80 ± 0.05

4±1

77 ± 1

0

5±1

2051 ± 0.05

2±1

70 ± 2

13 ± 2

22 ± 1 22 ± 1 20 ± 1

4.79 ± 0.05 5.25 ± 0.05 3.39 ± 0.05

4±1 4±1 4±1

68 ± 1 95 ± 1 54 ± 2

20 ± 4 0 0

4±1 4±1 20 ± 2

0.72 ± 0.04 1.79 ± 0.05 0024 ± 0.04

2±1 2±1 2±1

20 ± 2 50 ± 2 6±3

8±4 31 ± 4 9670 ± 150

20 ± 1

4.21 ± 0.05

4±1

68 ± 2

0

20 ± 2

0.41 ± 0.04

2±1

11 ± 4

6980 ± 100

22 ± 1

5.00 ± 0.05

4±1

90 ± 1

0

0±5

0 ± 0.04

2±1

0±1

lipid/NA (mol/mol)

none

a The hydrogenated surfactant/lipid was recorded in D2O and the deuterated surfactant in nrw. The errors are estimates based on the significance of the fits to the neutron reflectometry data in at least three different solvent contrasts.

ssRNA10 Alters the Domain Structure of DPPC Monolayers in the LE/LC Coexistence Region. We have now established that there is adsorption of nucleic acids to lipid/surfactant monolayers, and we aim to explore whether this also influences domain formation and morphology in the interfacial layer. We employed fluorescence microscopy combined with the monolayer equipment to study domain formation in the DPPC monolayers in the presence of ssRNA 10 , dsRNA 10 , and dsDNA 2000 . The experimental procedure was similar to that used in the surface balance Πtime studies. The monolayer was compressed to Πi = 11.0 ± 0.1 mN/m, where the LE and LC monolayer phases coexist and where well-defined domains are apparent (Figure 1b). Then, the nucleic acid was injected into the subphase. In Figure 5 representative images obtained for DPPC monolayers in the presence of the different nucleic acids are shown, as well as control experiment where only pure buffer solution was injected. The images were analyzed based on at least 40 images collected at randomly chosen positions in each monolayer, and this was done in several (2−4) experiments. The structures captured in the images were classified in terms of the different characteristic structures. In Figure 5a characteristic domains for the pure DPPC monolayer at Πi = 11.0 ± 0.1 are shown, where the LC phase is seen as dark islands in the bright surrounding LE phase. Upon addition of ssRNA10 to the subphase, the domains become interconnected and form a fractal-like structure, as shown in Figure 5b. The structures appear compact and no isolated domains with the characteristic shape as shown in Figure 5a can be distinguished. Almost identical fractal-like surface structures has previously been observed in a completely different lipid monolayer system composed of a crude lipid mixture extracted from myelin.42−44 The interconnected fractal-like structure is formed throughout the monolayer and only very few areas with discrete domains could be observed (in less than 5% of the images). The area covered by dark domains (LC phase) is ca. 20−25% in presence of ssRNA10, compared to ca. 10−15% LC domains for the neat DPPC monolayer where only pure buffer solution was injected to the subphase (Figure 5a). Figure 5c shows the dominating domain structures after the addition of dsRNA10. No fractal-like interconnected domains were observed, and the only difference compared to the neat DPPC monolayer (Figure 5a) is some distortion of the domain shape, and possibly also a

It is a striking observation that the addition of dsDNA2000 affects the monolayers in clearly different ways compared to ssRNA10. The scattering length density profiles of the DODAB monolayer spread on a subphase that contains dsDNA2000 together with fitted values suggest that dsDNA2000 partially remove the DODAB monolayer at both high and low Π. This effect is apparent when comparing the experimental data in the reflectivity curves, where the measured reflectivity is lower for the mixed DODAB+dsDNA2000 layer compared to the neat DODAB monolayer (Figure S2 and S3; d-DODAB, nrw contrast). The observed loss in reflectivity in the case of DNA adsorption indicates a reduction of deuterated material (in this case d-DODAB) at the interface. Still, the differences in the calculated values of volume fractions are very small, and the comparison between the neat monolayer and the DODAB +dsDNA2000 layer show that the content of DODAB in the layer decreases to from 58 ± 2 to 54 ± 2 vol % DODAB at Π = 8.8 ± 0.2 mN/m, and from 77 ± 2 to 68 ± 2 vol % DODAB at Π = 28.0 ± 0.2 mN/m (Tables 2 and 3). It is also noted that the addition of ssRNA10 indeed gives the opposite effect, where the reflectivity is higher for the mixed DODAB-ssRNA10 layer compared to the neat DODAB monolayer (Figure 4a). Based on the decrease in scattering length density, we can assume that no DNA is able to penetrate into the monolayer. The layer below the monolayer contains relatively little dsDNA2000, about 6−10 vol %. This corresponds to 7000− 9700 surfactants per dsDNA2000 molecule, which means that there is about 2-fold excess of surfactant charges compared to nucleic acid charges. The relatively large effect of dsDNA2000 adsorption on the measured Π (Figure 2) is likely not simply explained by the change in surface composition but is most likely due to rearrangement of the surfactants in the monolayer due to the presence of oppositely charged DNA. Finally, the NR data suggest that dsDNA2000 is not able to adhere to the DPPC monolayer under the given experimental conditions, at least not sufficiently to be detected by NR. In summary, the NR data show that ssRNA10 adsorbs and penetrates into LE monolayers at low Π (Πi = 8.4 ± 0.2 mN/ m), while there is no penetration into more condensed monolayers at higher Π (Πi = 8.4 ± 0.2 mN/m). Furthermore, no penetration of dsDNA2000 was observed for any systems investigated, and dsDNA2000 adsorption was only detected for the cationic DODAB monolayers. 9628


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ssDNA molecules used. Single stranded nucleic acids with up to several thousands of bases may, in spite of the fact that nucleic acids can be regarded as stiff polyelectrolytes, be able to bend and twist so that short segments of the strand may find complementary segments and thereby protect the hydrophobic bases from the aqueous surroundings.50 To avoid these uncertainties, we have chosen to use short RNA oligonucleotides with simple sequences. In this model system, we can capture the features of the interaction between exposed unpaired hydrophobic bases of RNA and model membranes. The 10 bp ssRNA10 oligonucleotides are composed of only one type of base (either polyA or polyU), to avoid self-association and back folding of the molecule. The dsRNA10 is composed of two complementary strands designed to avoid self-association and to avoid strand mismatching when pairing the two complementary strands. For the dsRNA10, the hydrophobic bases are shielded from contact with the aqueous solution due to base-pairing, whereas the hydrophobic bases are exposed in ssRNA’s. We also make the comparison with a longer polynucleic acid, that is the 2000 bp salmon sperm dsDNA2000, which has been used in several previous studies on similar systems.27,51 ssRNA10 Adsorbs Differently to LE and LC Monolayers. Figures 2−4 all demonstrate the significant differences in the adsorption of nucleic acids to the lipid/surfactant monolayers with a strong dependence on the base-pairing of the polynucleic acids. Our results can be explained by changes in the balance between electrostatic and hydrophobic interactions in the different systems. In the present study, we se no signs of adsorption of dsDNA2000 to zwitterionic DPPC monolayers. This is consistent with some previous studies on similar systems.27 The addition of ssRNA10, on the other hand, gives rise to an increase in Π both for the DPPC LE and the DPPC LE/LC monolayers, and a change in NR profile for the same systems. As none of the nucleic acids used are surface active on their own, the observed effects must be related to interactions with the lipid monolayer. Using NR reflectometry we were able to quantify the adsorption (Table 3), showing that a substantial amount of the ssRNA10 enters into the DPPC monolayer, and that ssRNA10 also is present below the headgroup region. The same conclusion could also be drawn for the system where ssRNA10 was added to the DODAB LE monolayer. The exposure of the hydrophobic bases of ssRNA might be the explanation to the large amount of ssRNA10 associated with the lipid/surfactant monolayer. In fact, the concentration of ssRNA10 in the monolayer corresponds to around three surfactant molecules per ssRNA10 for the DODAB LE monolayer (Table 3). This is far more ssRNA10 than would be expected if only the electrostatic attractive force was controlling the association. Hence, the driving force for association is likely partly due to the attractive interaction between the hydrophobic bases of ssRNA10 with the acyl chains of the monolayer, which is more exposed at the lower Π (Πi = 8.8 ± 0.2 mN/m2). From the NR reflectometry we were also able to observe that there is no penetration of ssRNA10 into the DODAB and DPPC monolayers when the monolayers are more condensed at high Π (Πi = 28.0 ± 0.2 mN/m2), where the hydrophobic lipid acyl chains are far less exposed. We also note that the concentration of ssRNA10 in the monolayer is lower for the zwitterionic DPPC LE monolayer compared to that of the corresponding cationic surfactant layer, whereas a relatively larger proportion of the adsorbed ssRNA10 penetrates

Figure 5. Fluorescence microscopy images of a DPPC monolayer with and without nucleic acids present in the subphase (Πi = 11 mN/m). The dark regions correspond to the LC phase and the bright regions to the LE phase. In panel b the effect of addition of ssRNA10 is clearly visible where the DPPC domains have formed interconnected structures. The addition of dsRNA10 gives a minor distortion of the DPPC domain structures, whereas the addition of buffer or of dsDNA2000 shows no significant change of domain structure.

decrease in domain size. The addition of dsDNA2000 gave almost identical results as the addition of pure buffer, and we could not observe any effects on the domain formation in any of these cases (Figure 5d). On a few occasions, the monolayer was disturbed during the injection process as implied from the observation that some domains formed thin threadlike structures. This structure has no resemblance with the more condensed fractal-like structure in Figure 5b.

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DISCUSSION RNA occurs more often than DNA in the single-stranded form, where the apolar parts of the bases are prone to hydrophobic interactions with other species. There are indeed many studies that aim to characterize the interaction between nucleic acids and other macromolecules, e.g., surfactants, lipids, and polymers.45,28,46−48 It is noticeable that, in some studies that aim to compare DNA association with surfactants or polymers, only small differences between single stranded and double stranded species are observed.49 One possible explanation to this might lie in the fact that it is difficult to control (or measure) the degree of exposed unpaired bases of the long 9629


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strands and the reforming of the molecule is likely relatively fast and the observed increase in Π may originate from the adsorption of free (or partially dissociated) single strands of RNA that might be present in the solution at 22 °C.53 Association of RNA and DNA to Cationic Monolayers. The adsorption behavior of the double-stranded and singlestranded species differs also for the cationic monolayers. We note that both dsDNA2000 and ssRNA10 adsorb to cationic DODAB monolayers, but only ssRNA10 penetrates into the monolayer. dsDNA2000 was only detected in the layer below the cationic headgroup region, and the association is likely purely driven by electrostatic attraction. These results are also consistent with previous monolayer studies on dsDNA interaction with cationic lipids/surfactants such as sphingosine and DODAB.54,51,28 The data in Figure 2 shows that the addition of the double-stranded nucleic acid species to DODAB monolayers gives rise to a larger increase in Π compared to that of the single-stranded ssRNA10. To justify this comparison, it is important to note that the concentration of charged bases is the same in all experiments. One plausible explanation for the observed differences is that molecules with higher charge density show stronger interactions to the oppositely charged monolayer, and the charge density of an extended single stranded RNA or DNA molecule is considerably lower than for a double stranded molecule.55 The strong association of dsDNA2000 with the DODAB monolayer is illustrated by the NR results, which imply that the dsDNA2000 even removes part of surfactant layer (Table 3). This effect is distinguished from the comparison to the monolayer on the neat buffer solution (Table 2). In this case, the large molecular size of the DNA molecule might be important. From the data presented in Table 3, we clearly see that there is an effect of the molecular size on the adsorbed amount as expressed in the lipid to surfactant molar ratio. The table shows that the amount of DNA is far from sufficient to compensate for the charges of the monolayer, which is different from the case of ssRNA10 at Πi = 28.0 ± 0.2 mN/m2, where the adsorbed amount matches the amount of surfactant in the monolayer on the basis of the charge ratio. The lower coverage of the dsDNA2000 can be attributed to the large repulsion in the interfacial plane, including both electrostatic and steric repulsion between adsorbed molecules. Another interesting observation is that the addition of ssRNA10 to the DODAB monolayer gives rises to an anomalous dip in ΔΠ immediate after the injection, whereas no such effects were observed for any of the double-stranded moieties. It is possible that this can be related to the exposure of the hydrophobic bases in the ssRNA10, which gives rise to additional hydrophobic attraction to the acyl-chain of the DODAB molecules. It is indeed possible that DODAB associate with the ssRNA10 forming a complex that is soluble in the subphase, thus removing material from the surface. This is, however, not observed in the NR study. Here we note that the time resolution is not sufficient to capture any dip in the NR measurements. In particular as we in the NR measurement performed the study with the surfactant spread on the subphase that contain the nucleic acid rather then injecting the nucleic acid below the monolayer. ssRNA10 Alters the DPPC Domain Structure. The nucleic acids do not only adsorb to the monolayers but were also shown to affect the LE/LC domain formation in some systems (Figure 5). The addition of ssRNA10 to DPPC LE/LC monolayers has a significant effect on the domain morphology and it leads to the formation of fractal-like interconnected

in the DPPC LE monolayer. Finally, the results from the NR studies (Table 3) can be related to the results summarized in Figure 6, which compares the effects on ΔΠ caused by the

Figure 6. ΔΠ vs time measurements comparing the adsorption of ssRNA10 to DPPC monolayers at different Πi with DODAB at Πi = 9 mN/m. Injection of ssRNA10 gives an increase in ΔΠ for DPPC monolayers where the LE phase is present and for the DODAB monolayer. For DPPC monolayer in LC phase, no increase in ΔΠ was ever observed.

addition of ssRNA10 to monolayers in different phases and with different composition. It shows that ssRNA10 affects both zwitterionic and cationic LE monolayers, whereas we cannot detect any effect on the DPPC LC monolayer. dsRNA 10 and dsDNA 2000 Do Not Interact with Zwitterionic Monolayers. It is an important conclusion that the adsorption of the double-stranded species to the zwitterionic monolayers is clearly different from the adsorption of the single-stranded ssRNA10 to the same monolayers. From the results obtained by means of Langmuir surface balance (Figure 3) and NR (Table 3 and SI), we note that there is no significant association of the double-stranded nucleic acids, dsDNA2000 and dsRNA10, to the DPPC monolayer under the present solution conditions. These results can be explained in that the double-stranded nucleic acids do not expose their hydrophobic bases and are thus not prone to hydrophobic interactions. These results are in agreement with previous studies on dsDNA2000 and zwitterionic monolayers in the absence of divalent cations.51,27 It can also be compared to the results presented by Cardenas et al. showing significant adsorption of a 100 bases ssDNA to hydrophobic surfaces, but only minor adsorption of dsDNA, independent of molecular size.32 However, there are also studies showing minor adsorption of dsDNA2000 to both gel and liquid crystalline zwitterionic bilayers in the absence as well as in the presence of divalent cations.30,52 The discrepancy between these studies and the present work might be assigned to the differences in solution conditions (salt concentration and pH), choice of model system (monolayers or bilayers), and the sensitivity of different techniques to very subtle effects. Finally, the data in Figure 3 implies that the short dsRNA10 adsorbs to zwitterionic monolayers to some extent. One plausible explanation to the observed association is the low denaturation temperature of the short dsRNA10 helices (26 °C, as stated by the manufacture). The dissociation of the dsRNA10 into single 9630


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domain structures. We speculate that the ssRNA 10 is preferentially associated to the LE domains and possibly excluded from the LC domains. Such an arrangement could lead to the growth of the LE domains and concomitant contraction of the LC domains. It is also possible that ssRNA10 might act as a line active (cf. surface active) agent between the two-dimensional liquid expanded and liquid condensed phase and thus reducing the line tension along the domain boundaries. Line active properties have previously been reported for, e.g., cholesterol and some proteins.56,57 The observed increase in the total area covered by LC domains can likely be explained by the increased Π after the adsorption of ssRNA10. Fractal like patterns in lipid monolayers have also been reported by Oliviera et al. as well as by Rosetti et al. which shows that the domain structures of myelin monolayers formed foam-like surface structures in the presence of a Folch-Lees proteolipid protein.42−44 The proposed explanation for these observations was that the fractal-like domains were formed due to inhomogeneity of the surface composition and lateral segregation of the components. However, the authors point out that the molecular cause for formation of the specific fractal-like domain shapes is yet unknown.

■

CONCLUSIONS Biological membranes are to a large extent built up by phospholipids, and PC is one of the most abundant types of phospholipids in for example the plasma membrane.58−60 As the interest of using, e.g., miRNA in therapeutics for treatment of various diseases increases,3,4,12 there is a need to develop a basic understanding of the interaction between these types of molecules and lipids. In pharmaceutical research, zwitterionic phospholipids have for example been of interest to use as delivery vehicles due to the low cytotoxicity. In this study, we have characterized the association of single-stranded and double-stranded nucleic acid species with zwitterionic and cationic monolayers with different phase behavior by means of surface balance measurements, neutron reflectometry, and fluorescence microscopy. The major conclusions are as follows: • Short single-stranded ssRNA10 associates with zwitterionic phospholipid liquid expanded monolayers but not with liquid condensed monolayers. We propose that the association is driven by the hydrophobic interactions between the exposed hydrophobic bases of the ssRNA10 and the exposed hydrocarbon chains of the phospholipid in the expanded monolayer. • The NR studies demonstrate that ssRNA10 penetrates into expanded monolayers composed of zwitterionic DPPC or cationic DODAB. On the other hand, no penetration is detected for the more condensed monolayers of the same composition. This implies that the lipid phase behavior is crucial to the interaction and that the association is a consequence of both attractive electrostatic interactions and hydrophobic interactions. • In the comparison between different nucleic acid systems, we conclude that the degree of base pairing as well as the molecular size influences the association behavior. The double-stranded species dsDNA2000 and dsRNA10 show no significant association with zwitterionic monolayers. Furthermore, no penetration of dsDNA2000 into the monolayer could be detected by means of NR for any system investigated. This can be explained by the fact that the double-stranded species do

■

not expose their hydrophobic bases and are therefore less prone to hydrophobic interactions with the apolar region of the monolayer. • The ssRNA 10 significantly influences the domain morphology in the LE/LC DPPC monolayer, where it leads to the formation of fractal-like interconnected domain structures. Similar effects on domain morphology were not observed for any of the double-stranded species. We speculate that the observed effects can be related to line-active properties of ssRNA10. • In this work we have established the importance of the hydrophobic interactions between single-stranded nucleic acids and fluid model membranes. Our data was recorded for expanded lipid monolayers, and this model system is relevant for biological membranes and fluid lipid bilayer where there can be significant exposure of hydrophobic acyl chains.61 The findings are relevant for the understanding of uptake and transport of nucleic acids in living systems and in drug delivery vehicles. One important message is that the association of nucleic acids with amphiphiles is not only controlled by electrostatic attraction but that also hydrophobic interactions can play an important role in the case of single stranded nucleic acid species. This could be exploited in the design of new drug delivery systems for gene therapy.

ASSOCIATED CONTENT

S Supporting Information *

Additional data recorded for other isotope contrasts as well as data recorded for the same systems at Πi = 28.0 ± 0.2 mN/m and for the same monolayer systems in the presence of dsDNA2000 and corresponding profiles for other isotopic contrasts. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors thank Christian Gudmundsen for help with monolayer measurements. The Swedish Research Council (VR) is gratefully acknowledged for financial support both through regular grants and the Linnaeus Center of Excellence “Organizing molecular matter”. E.S. acknowledges The Swedish Foundation for Strategic Research (SSF) for financial support. Per-Eric and Ulla Schyberg’s foundation and the Crafoord foundation funded the acquisition of the monolayer equipment.

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REFERENCES

(1) Ambros, V.; Chen, X. M. The regulation of genes and genomes by small RNAs. Development 2007, 134, 1635−1641. (2) Bartel, D. P. MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell 2004, 116, 281−297. (3) Ambros, V. The functions of animal microRNAs. Nature 2004, 431, 350−355. (4) Juliano, R.; Alam, M. R.; Dixit, V.; Kang, H. Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Res. 2008, 36, 4158−4171. (5) Meltzer, P. S. Cancer genomics - Small RNAs with big impacts. Nature 2005, 435, 745−746. (6) Garzon, R.; Calin, G. A.; Croce, C. M. MicroRNAs in Cancer. Ann. Rev. Med. 2009, 60, 167−179.

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RNA and DNA Association to Zwitterionic and Charged Monolayers at

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