Characterization of a Lipophilic Plasmid DNA Condensate Formed

ARTICLE pubs.acs.org/Biomac

Characterization of a Lipophilic Plasmid DNA Condensate Formed with a Cationic Peptide Fatty Acid Conjugate Trinh T. Do,† Vicky J. Tang,† Joe A. Aguilera,† Christopher C. Perry,‡ and Jamie R. Milligan*,† † ‡

Department of Radiology, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093-0610, United States Department of Biochemistry, Loma Linda University, 11085 Campus Street, Loma Linda, California 92350, United States

bS Supporting Information ABSTRACT: In the presence of cationic ligands, DNA molecules can become aggregated into larger particles in a process known as condensation. DNA condensates are of interest as models for the dense packing found in naturally occurring structures such as phage heads and chromatin. They have found extensive application in DNA transfection and also provide convenient models with which to study DNA damage by the direct effect of ionizing radiation. Further, conjugates of cationic peptides with fatty acids may represent a class of attractive ligands for these areas because of their simple synthesis. When plasmid pUC18 is used as the DNA target and N-caproyl-penta-arginine amide (Cap-R5-NH2) is used as the ligand, the physical properties of the resulting mixtures were characterized using static and dynamic light scattering, sedimentation, dye exclusion, circular dichroism, nanoparticle tracking, and atomic force microscopy. Their chemical properties were assayed using solvent extraction and protection against hydroxyl radical attack and nuclease digestion. Titration of the plasmid with the Cap-R5-NH2 ligand produced sharply defined changes in both chemical and physical properties, which was associated with the formation of condensed DNA particles in the 100
2000 nm size range. The caproyl group at the ligand’s N-terminus produced a large increase in the partitioning of the resulting condensate from water into chloroform and in its binding to the neutral detergent Pluronic F-127. Both the physical and chemical data were all consistent with condensation of the plasmid by the ligand where the presence in the ligand of the caproyl group conferred an extensive lipophilic character upon the condensate.

’ INTRODUCTION The phenomenon of DNA condensation1 has been for several decades a subject of study for two main reasons. First, it offers the ability to study the energetics of the highly compact organization of DNA found in naturally occurring structures including phage heads, viral capsids, and eukaryotic chromosomes.2,3 DNA molecules, particularly so in the nuclei of cells, are in nature extensively compacted into volumes with dimensions orders of magnitude smaller than their overall lengths.3 Second, DNA compaction has played a central role in the development of delivery systems for gene therapy that avoid the limitations of viral transduction.4 Moreover, DNA compaction has been found to increase the efficiency of membrane penetration and subsequent expression.5 The mechanism by which condensation takes place may include compaction and aggregation. In compaction, a single DNA molecule will occupy a smaller volume, while, in aggregation, multiple molecules become associated with one another. Because DNA is an anionic polymer, these processes are characterized by a substantial electrostatic barrier.1 It is found that DNA condensing agents are in many cases cationic species such as the complex ion hexa-ammine cobalt(III),6 polyamines such as spermidine and spermine7 and derivatives of them,8 cationic (viz. basic) proteins such as nucleosomes and protamines,9 and artificial polymers such as polyethylene imine.10,11 r 2011 American Chemical Society

The compact arrangement of DNA found in a phage or virus is at a concentration of about 800 mg mL
1, while a value of about 170 mg mL
1 is representative of a mammalian metaphase chromosome.3 Condensed DNA is able to achieve packing densities of this order. For example, the distance between adjacent double helices in condensed DNA has been reported as 2
3 nm,12 a value typical of the super helical pitch of the nucleosome.13 Condensed DNA has applications in the study of DNA damage by ionizing radiation.14 It provides a suitable model15 with which to examine the relative contributions made to DNA damage by the direct and indirect effects of ionizing radiation.16,17 The direct effect is the route by which chemical modification of the DNA is produced by ionization of the DNA itself. In the indirect effect, ionization of nearby solvent molecules produces highly reactive radical intermediates. These species tend to react on encounter but may diffuse distances of a few nanometers before reacting with and chemically modifying the DNA. These two reactions may be distinguished from one another because the indirect effect can be attenuated by Received: January 26, 2011 Revised: March 1, 2011 Published: March 16, 2011 1731

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Biomacromolecules competing for the diffusible intermediates with added scavengers. In contrast, no additive can interfere with the introduction of damage by the direct effect (although they may certainly affect the subsequent chemical reactions). The efficiency of the direct effect in biological systems is assumed to be equal to the fraction of the ionizable electrons contributed by the DNA. For low atomic number systems such as those found in biology, this is to a good approximation the fraction of the mass. For mammalian cells, the overall DNA content is about 0.1% by mass, implying that the direct effect makes only a minor contribution. However, the chromosomal concentration value of 170 mg mL
1 quoted above suggests that the direct effect may well be much more efficient. Indeed, observations with very high concentrations of nontoxic cryoprotectants such as glycerol and dimethyl sulfoxide18 and sophisticated biophysical models of radiation damage19 both agree that the contribution of the direct effect to DNA damage in cells is over 2 orders of magnitude greater than the 0.1% value and in general consistent with the 170 mg mL
1 value. The interpretation is that the effectiveness of the direct effect is enhanced by the very high packing density of DNA and that the indirect effect is very strongly attenuated by scavenging of solvent derived radical intermediates in the immediate neighborhood of the genomic DNA in a cell nucleus. Thus, the high DNA concentrations and close physical association of DNA binding proteins are essential to understanding the large contribution made by the direct effect to DNA damage in biological systems. This effect becomes even more important for the more densely ionizing forms of radiation (described as having a high linear energy transfer, or high LET).16 Examples of this are low energy X-rays and particulate radiations such as neutrons, alpha particles, and fission fragments. Biochemical model systems for ionizing radiation damage of DNA which consist of dilute aqueous solutions are good models for the indirect effect, because reactions of solvent derived radicals are dominant. They are however poor models for the direct effect. It is necessary to decrease the efficiency of the indirect effect by several orders of magnitude before the effects of the direct effect become detectable. Experimental methods to achieve this typically involve very high (molar) concentrations of scavengers, dehydration,20,21 and cryogenic temperatures.22,23 The purpose is, respectively, to intercept, prevent the formation, or prevent the diffusion of the intermediates responsible for the indirect effect. Such conditions are not unusual in physical chemistry, but are considered extreme by the standards of biophysics and biochemistry. Therefore, attention turns to the possibility of exploiting condensed DNA as a means to model the direct effect in an otherwise dilute aqueous solution. Several advantages are apparent. Condensation produces a large decrease in the efficiency of the indirect effect under mild conditions in a room temperature aqueous solution.24 It is easily produced and reversed by minor alterations in the composition of the sample, such as the ionic strength.24
26 A wide range of condensing agents is available (see above). The radioprotective effects of polyamines have been studied.27
29 We have argued that small cationic oligopeptides represent convenient ligands with which to study the chemical reactions that take place after DNA damage by the direct effect because they are able to act as DNA condensing agents.24,30 Electron transfer reactions with amino acid residues such as tryptophan and tyrosine appear to play an important role.31 These amino

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acids can be conveniently tethered to cationic ligands by incorporating them into small DNA binding25,32 peptides rich in basic amino acid residues such as arginine and lysine. An extension of this approach involves incorporating fatty acids into the cationic ligand,14 which appears to further limit the accessibility of water to reactive intermediates located in the DNA. Applications include modeling solvent and membrane effects on DNA damage33
35 and transfection efficiencies.10

’ EXPERIMENTAL SECTION Sources of Biochemical Reagents. A sample of the plasmid pUC18 was obtained commercially (Invitrogen, Carlsbad, CA). It was grown in transformed bacterial cells on a milligram scale, isolated, and purified as described previously.36 The ligands penta-arginine (R5) and N-caproyl-penta-arginine amide (Cap-R5-NH2) were obtained commercially (BioMatik, Wilmington, DE, and Biosynthesis, Lewisville, TX, respectively). Recombinant bovine pancreatic DNase I was obtained from Roche (Indianapolis, IN). Composition of Plasmid Solutions. Various assays described below were applied to aqueous solutions containing sodium phosphate (5  10
3 mol L
1, pH 7.0), plasmid pUC18 (10 μg mL
1, equivalent to a base pair concentration of 1.5  10
5 mol L
1), the ligand Cap-R5-NH2 or in some cases its parent R5 (zero, or 1  10
6 to 8  10
5 mol L
1), and in some cases the detergent Pluronic F-127 (zero to 0.1% w/v). Because in many cases the physical properties of mixtures of plasmid DNA and ligands changed for some minutes after mixing, in most cases the assays described below were made after waiting 30 min after addition of the ligand. Similar effects have been reported in the literature.37,38 Static Light Scattering. The intensity of static light scattering at a nominal angle of 90° was quantified (200 μL aliquot) using a model F-7000 fluorescence spectrophotometer (Hitachi, Pleasanton, CA) with both excitation and emission monochromators set to 350 nm (bandwidth in both cases 5 nm). Sedimentation and Solvent Extraction. The fraction of the plasmid remaining in solution after centrifugation at 15,000  g for 10 min (150 μL aliquot) was determined from the absorbance at 260 nm (bandwidth 1 nm) of the supernatant (aliquot of 120 μL). This method was also used to quantify the fraction of the plasmid remaining in the aqueous upper phase after extraction with an equal volume of chloroform (aliquot of 1 mL). These measurements employed a model DU800 spectrophotometer (Beckman Coulter, Fullerton, CA). Ethidium Fluorescence. The displacement of ethidium (at a final concentration of 10
6 mol L
1) from the plasmid was assayed (200 μL aliquot) using the decrease in fluorescence with excitation at 510 nm (bandwidth 5 nm) and emission at 590 nm (bandwidth 5 nm) using a model F-7000 fluorescence spectrophotomer (Hitachi, Pleasanton, CA). The anisotropy of bound ethidium was also quantified under these conditions.39 Gamma Irradiation. The sensitivity of plasmid DNA in the additional presence of 10
4 mol L
1 glycerol to degradation by the hydroxyl radical was assayed (18 μL aliquot) with cesium-137 γradiation (662 keV) using a GammaCell-1000 instrument (J. L. Shepherd, San Fernando, CA). The dose rate of 288 rad min
1 was calibrated using the Fricke system16 and with commercial thermoluminescent and optically stimulated luminescent dosimeters (Landauer, Glenwood, IL). The fraction of intact supercoiled plasmid remaining after irradiation was determined using agarose gel electrophoresis. The resulting yield of DNA single-strand breaks was quantified as described previously.36 This radiation chemical yield has units of moles of SSB product per joule of energy deposited by the gamma ray photons. Nuclease Digestion. The sensitivity of plasmid DNA to digestion by DNase I was involved in a 60 min incubation at 37 °C in the additional presence of 10
4 mol L
1 magnesium chloride and 10
1 to 10
3 units μL
1 1732

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Biomacromolecules of DNase I. The reaction (16 μL) was halted by addition of EDTA (2 μL) to a final concentration of 2  10
2 mol L
1. The fraction of intact supercoiled plasmid was determined using agarose gel electrophoresis. The efficiency of DNA single-strand break formation by DNase I was quantified from the slope of a plot of the number of SSB events per plasmid (assuming a Poisson distribution this is equal to the natural logarithm of the reciprocal of the fraction of the plasmid remaining in the supercoiled form) against the enzyme concentration. This efficiency was quantified as SSBs per plasmid per unit activity of the enzyme per mL. Agarose Gel Electrophoresis. Immediately prior to loading the sample aliquots (18 μL) of nuclease digested or gamma irradiated plasmid were treated with a loading buffer (4 μL) containing sodium perchlorate (1 mol L
1), sucrose (40%), and bromophenol blue (1 mg mL
1). The resulting solutions were subjected to electrophoresis using a 1.3% agarose gel in the TBE buffer system at 55 V for 13 h. The fraction of the plasmid in the supercoiled form was quantified by digital video imaging with a Gel Doc XR instrument (Bio-Rad, Hercules, CA) of the ethidium fluorescence of its supercoiled, open circle and (if present) linear forms. UV
Visible Spectroscopy. Absorption spectra of mixtures of plasmid DNA and the ligand Cap-R5-NH2 were recorded using a model DU-800 spectrophotometer (Beckman Coulter, Fullerton, CA) under the following conditions: path length 1 cm, scan speed 1200 nm min
1, resolution 1 nm. Circular Dichroism Spectroscopy. The CD spectrum of plasmid DNA was recorded using a model J-715 spectropolarimeter (Jasco, Easton, MD) in the presence of either the penta-arginine ligand or hexaammine cobalt(III) ions. In each case, eight scans were accumulated under the following conditions: path length 2 mm, scan speed 100 nm min
1, resolution 0.2 nm, response time 1 s. The spectra were corrected for the cuvette and buffer signals but were not subjected to any smoothing procedures. Dynamic Light Scattering. The size range of plasmid
ligand condensates was quantified (3 mL aliquot) by dynamic light scattering using a model 370 instrument (Nicomp, Santa Barbara, CA). The illumination source was a 60 mW helium
neon laser operating at 632 nm. Data was collected at a scattering angle of 90° and fitted with a proprietary autocorrelation function to calculate the distribution of the diffusion coefficient. The apparent spherical hydrodynamic radius was then estimated assuming the Stokes
Einstein equation. Nanoparticle Tracking. The size range of plasmid
ligand condensates was also quantified (500 μL aliquot) by the technique of nanoparticle tracking analysis40,41 using a model LM20 instrument (Nanosight, Costa Mesa, CA). This involved microscope video capture (640  480 pixels, 30 frames per second) of the light scattered by individual particles and a subsequent size estimate derived from their Brownian motion using proprietary software, again based on the Stokes
Einstein equation. Atomic Force Microscopy. Samples were prepared by depositing a 5 μL aliquot onto a surface of freshly cleaved mica (1  1 cm), followed by air drying. The samples were not deposited in the presence of any additional cations (such as the commonly employed Mg2þ). Images were collected using a Dimension 5000 instrument (Veeco, Santa Barbara, CA) operated in the tapping mode. The cantilevers (Arrow NCR-50, Nanoworld, Neuchatel, Switzerland) were 160 μm long, with a resonant frequency of 285 kHz. The scan speed was 1 line of 256 pixels per second. The image is topographic (intensity corresponds to height). Minor image processing was used to remove horizontal scanning artifacts and to improve contrast.

’ RESULTS AND DISCUSSION A wide variety of oligocationic ligands bind electrostatically to the polyanionic DNA. The resulting extensive charge neutralization can

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Figure 1. Panel A (square symbols): effect of the concentration of the ligand Cap-R5-NH2 on light scattering by plasmid pUC18. The relative intensity of light (wavelength 350 nm, bandwidth 2 nm) scattered at 90° was quantified using a fluorescence spectrometer. Panel B (circular symbols): effect of the concentration of the ligand Cap-R5-NH2 on the sedimentation of plasmid pUC18. After centrifugation, the plasmid concentration in the supernatant fraction was estimated from its UV absorbance.

in some cases promote a collapse of the DNA into a more compact form and its aggregation into particles with sizes in the range of hundreds of nanometers.1 This process is referred to as DNA condensation. It provides a model system for the highly compacted DNA found in naturally occurring structures such as phage heads and chromosomes3 and has found an important technological application in improving the efficiency of DNA transfection.4,42 We wished to characterize the ability of the C-8 fatty acid peptide conjugate Cap-R5-NH2 to act as a DNA condensing agent and to compare this ability to that of its peptide parent penta-arginine (R5, which we have examined previously). The carboxylic amide of the fatty acid conjugate was used so that it has the same overall charge (Z = þ5) as penta-arginine. This minimizes any charge related effects, since ligand charge is well-known to play an important role in DNA binding and condensation.25 Using a variety of analytical methods, we found consistent evidence that Cap-R5-NH2 is able to act as a DNA condensing agent. Static Light Scattering and Sedimentation. The effect of the ligand Cap-R5-NH2 on pUC18 plasmid DNA was examined by using static light scattering through 90° at a wavelength of 350 nm. Figure 1A shows the dependence of the relative intensity of scattering on the ligand concentration. We observed an increase in intensity of about 10-fold over a narrow concentration range. The midpoint of this effect is located at a ligand concentration of about 7  10
6 mol L
1. Very similar effects have been reported with structurally closely related ligands such as tetraand penta-lysines31 and with penta-arginine.15 The wavelength dependence of scattering was also examined over the range 200
800 nm (Supporting Information, Figure 1). A typical DNA absorption is visible below 300 nm, and scattering is evident as an increase in the background, which becomes more pronounced at shorter wavelengths. Mixtures of the plasmid and Cap-R5-NH2 were also subjected to a brief centrifugation. The fraction of the plasmid remaining in solution was quantified by UV spectrophotometry and is plotted 1733

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Figure 2. Effect of the concentration of the ligand Cap-R5-NH2 on the fluorescence intensity (panel A, square symbols) and fluorescence anisotropy (panel B, circular symbols) of plasmid-bound ethidium. Ethidium fluorescence (excitation 510 nm, emission 590 nm, bandwidth in both cases 5 nm) was quantified using polarizing filters in both the excitation and the emission light paths.

against the ligand concentration in Figure 1B. We observed a modest decrease of ca. 25% in the plasmid concentration at ligand concentrations in the range of 3  10
6 to 6  10
6 mol L
1, and a sharp decrease to undetectably low values at ligand concentrations greater than 7.5  10
6 mol L
1. As above, similar effects have been reported with penta-lysine and pentaarginine. These increases in static light scattering and sedimentation both provide strong evidence that multiple plasmids are aggregated by the ligand into much larger particles. Ethidium Fluorescence. The intercalating dye ethidium finds frequent use as a DNA probe.39 A decrease in ethidium fluorescence is generally assumed to derive from a decrease in the fraction of the dye which is bound to the plasmid.43 It was prebound to the plasmid at a level of 1/15th that of the base pair concentration. The intensity and the anisotropy of its fluorescence (see Figure 2) were both monitored as a function of the Cap-R5-NH2 concentration under conditions otherwise identical to those used above for static light scattering and sedimentation. The relative intensity of fluorescence due to ethidium decreased by about 4-fold in a roughly sigmoidal manner over a ligand concentration range of 2  10
6 to 2  10
5 mol L
1. The midpoint of this effect is located at a ligand concentration of about 6  10
6 mol L
1. The fluorescence anisotropy increased from about 0.10 to about 0.15 over the same concentration range. A large change in response over a very small concentration range argues against a simple binding competition between the ligand and the dye and is instead suggestive of an altered conformation to which ethidium binds significantly less strongly. The increase in anisotropy of the fluorescence due to the bound eithidium implies that the residual dye is less mobile and that the plasmid experiences a limited flexibility when bound into aggregates. The DNA condensation that is observed over a narrowly defined ligand concentration of ca. 6  10
6 mol L
1 with a charge of Z = þ5 on the ligand corresponds to an ability to neutralize a base pair concentration of (5/2)  6  10
6 = 1.5  10
5 mol L
1.

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Figure 3. Panel A (square symbols): effect of the concentration of the ligand Cap-R5-NH2 on the SSB yield caused by gamma irradiation. The SSB yield was quantified from the fraction of the supercoiled form of the plasmid remaining after various doses of gamma irradiation. Panel B (circular symbols): effect of the concentration of the ligand Cap-R5-NH2 on the number of SSBs produced in plasmid pUC18 by DNase I. The number of SSB events per plasmid per unit per mL was quantified using dilutions of the enzyme DNase I.

This is comparable to the concentration of the plasmid. Because DNA condensation typically requires the neutralization of about 90% of its negative charges,1 this close agreement between cation and anion concentrations implies a near quantitative binding of the ligand under the experimental conditions. Therefore the presence of the C-8 caproyl group in the ligand does not appear to inhibit its DNA binding ability. Radiation Damage and Nuclease Digestion. We also examined the effect of the presence of the ligand Cap-R5-NH2 on access to the plasmid by two different reagents, both of which irreversibly modify its deoxyribose-phosphate backbone. These were the hydroxyl radical and the enzyme DNase I. The hydroxyl radical was generated by exposing the solution to ionizing radiation under aerobic conditions identical to those employed above, except that 1  10
4 mol L
1 glycerol was additionally present to overwhelm any scavenging effects of the ligand. The radiation chemical yield of single strand breaks (SSBs) introduced into the plasmid under these conditions is plotted in Figure 3A against the ligand concentration. The break yield decreases by about 100-fold over a narrow range centered at about 6  10
6 mol L
1 of the ligand. The sensitivity of the plasmid to SSB formation by the nuclease DNase I was quantified in a similar manner under conditions identical to those described above for static light scattering and sedimentation, except for the additional presence of 1  10
4 mol L
1 magnesium ions required as a cofactor for the enzyme. The efficiency of SSB formation was found to decrease by about 50-fold over a narrow ligand concentration range centered at 6  10
6 mol L
1 (Figure 3B). Strand break formation in plasmid DNA by ionizing irradiation under the poorly scavenged (10
4 mol L
1 glycerol) conditions we employed is caused by the reaction of the hydroxyl radical with the deoxyribose groups.44 The large attenuation observed in the radiation sensitivity of the plasmid argues that in 1734

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Figure 4. Circular dichroism spectroscopy of plasmid pUC18 in the presence of the ligand Cap-R5-NH2 (zero, 1  10
6, 2  10
6, 5  10
6, 8  10
6, or 1  10
5 mol L
1, panel A) or the ligand hexaammine cobalt(III) (zero, 5  10
6, 1  10
5, or 2  10
5 mol L
1, panel B). In both cases, higher concentrations are indicated with darker lines, and the direction of increasing concentration is indicated with an arrow.

the aggregated form it experiences an extensive protection against the hydroxyl radical, a small diatomic reagent. A ligand concentration of less than 10
5 mol L
1 is unable to compete with 10
4 mol L
1 glycerol as a hydroxyl radical scavenger,45 and in any case, a competition mechanism would produce a gradual decrease in strand break yield, which is quite different from the sharp decrease evident in Figure 3A. A physical protection of the plasmid aggregates from the bulk of the solution appears to be responsible. A similarly large protection was also found using bovine pancreatic DNase I. This much bulkier reagent (molecular weight 37 kDa) catalyzes the hydrolysis of phosphate diester groups in DNA. The resistance of the condensed plasmid to its nuclease activity is also consistent with a physical protection of the large DNA aggregates. All of these observations are consistent with the condensation of the plasmid by the ligand. Other commonly used ligands that produce similar light scattering, sedimentation, ethidium binding, and nuclease protection effects include the polyamines spermidine and spermine6 and the inorganic complex ion hexaammine cobalt(III).46 Radioprotective effects have been observed with oligolysine and oligoarginine peptides.24,31 Circular Dichroism Spectroscopy. Conformational effects of binding of Cap-R5-NH2 to the DNA helix were examined under the same conditions used for the light scattering and sedimentation results. To do so, we examined the circular dichroism (CD) spectrum in the 200
350 nm region (Figure 4A) and compared the results to those produced by the well-studied ligand hexaammine cobalt(III) (Figure 4B). In the absence of any added ligand, the CD spectrum is typical of the B-form of DNA with positive bands at about 220 and 270 nm. Concentrations of CapR5-NH2 too low to produce any scattering or sedimentation effects drive these bands in the negative direction but do not change their location. Higher ligand concentrations that do result in changes in light scattering and sedimentation behavior also generate large negative CD bands at 220 and 260 nm. These intense CD signals are characteristic of DNA condensation.47 In

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contrast, hexa-ammine cobalt(III) produces relatively minor changes in the CD spectrum of DNA (Figure 4B). These observations are consistent with literature reports.48 Time Resolved Coherent Light Scattering. The light scattering and sedimentation data in Figure 1 strongly suggest that the ligand Cap-R5-NH2 was acting to aggregate the plasmid into larger particles. We employed two different time-resolved assays to estimate the size range of these particles and to assess their interaction with a neutral detergent, the triblock copolymer Pluronic F-127.49 One of these was traditional dynamic light scattering (DLS)50 and the other was the more recently developed nanoparticle tracking analysis (NTA).40 The latter method involved viewing a laser illuminated suspension through a microscope objective and software tracking in real time of the Brownian motion of multiple individual particles. At a Cap-R5-NH2 concentration of 5  10
6 mol L
1, which is at a concentration too low to produce any aggregation, the hydrodynamic radius of the plasmid was found by DLS to be 110 ( 13 nm in the absence of Pluronic F-127 and 90 ( 13 nm in the presence of 0.1% Pluronic F-127 (Supporting Information, Figure 2). The corresponding radii at the 2-fold larger ligand concentration of 1  10
5 mol L
1, where aggregation occurs are, respectively, 1030 ( 120 nm (9-fold greater) and 1620 ( 210 nm (18-fold greater). In all cases, the size distributions as assayed by DLS were essentially monodisperse (>97%) and there was no significant difference between the intensity and volume weighted estimates of the hydrodynamic radius (Supporting Information, Figure 2). Very different results were found by applying the NTA assay to these same samples. First, no particles were observed at ligand concentrations below 5  10
6 mol L
1. Second, at ligand concentrations above 1  10
5 mol L
1, where Figure 1 reveals a large increase in static light scattering and in sedimentation rate, the light scattering particles observed had a heterogeneous size distribution. Third, because of the rapidity of this technique, it was possible to observe the dynamics of particle condensation at the higher ligand concentration during the time course of 10 to 15 min after addition of the ligand. The additional presence of Pluronic F-127 had no detectable effect on the broad size distributions or on the rate of aggregation. The 10th, 50th, and 90th percentiles of the size distributions are plotted against time in Figure 5. In attempting to quantify the size range of the aggregated particles in solution, these two light scattering assays produced mutually inconsistent results. DLS reported in all cases a monodisperse and fairly narrow distribution of particle sizes. It suggested that the plasmid was condensed from individual monomeric particles with sizes of about 100 nm into much larger particles with sizes in the micrometer range. In contrast, NTA was unable to detect individual uncondensed plasmids, but found the condensates to contain a very broad size distribution of particle sizes with dimensions from 200 nm to over a micrometer. The inconsistencies between these two assays of Brownian motion are a reflection of their limitations and provide further evidence that both should be interpreted with caution. Examples of these limitations include the inherent high sensitivity of DLS to small numbers of large particles, because of the sixth power dependence of the scattering intensity;41 the possibility of multiple scattering in DLS; the assumptions involved in deriving the diffusion coefficient from the DLS autocorrelation function; and that the finite number of particles examined by NTA might not constitute a representative sample. Detailed comparisons of the two methods can be found in the literature.41,51
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Figure 5. Hydrodynamic radius distributions of plasmid pUC18 as estimated by nanoparticle tracking. The plasmid was condensed with 1  10
5 mol L
1 Cap-R5-NH2 in the presence of either zero (open circle) or 0.1% w/v (closed circle) Pluronic F-127. The lowest decile, median, and highest decile of the size distribution are plotted as a function of time. Adjacent data points in each data set are linked with broken (open circle) or solid (closed circle) straight line segments. Figure 7. Panel A: representative atomic force microscopy image of plasmid pUC18 in the presence of 1  10
5 mol L
1 Cap-R5-NH2 and 0.1% Pluronic F-127 (panel A). The dimensions of this image are 20  20 μm in the horizontal plane. The vertical scale is 0.5 μm. Larger distances in the vertical axis above the plane are indicated with lighter pixels. Panel B: an image of 0.1% Pluronic F-127 alone at the same scale.

Figure 6. Partitioning of plasmid DNA between chloroform and water. Plasmid pUC18 DNA was treated with either R5 (open square) or CapR5-NH2 (closed square). The resulting solution was extracted with chloroform, and the plasmid content of the aqueous phase was quantified by its UV absorbance.

Solvent Partitioning. The lipophilic effect of the C-8 caprylate group in Cap-R5-NH2 was assayed by quantifying the partitioning of the plasmid between aqueous solution and an organic solvent. For control purposes, we compared the effect of the parent penta-arginine (R5). Thus, a mixture of plasmid DNA with either R5 or Cap-R5-NH2 was extracted with chloroform, and the residual DNA content of the aqueous phase was determined by UV spectrophotometry. The fraction of the plasmid remaining in the aqueous phase was plotted against the concentration of the ligands (Figure 6). Although changes in light scattering and sedimentation showed a very similar concentration dependence for these two ligands, as described above,15 the lipophilicity was very different. The majority of

the plasmid remained in the aqueous phase when R5 was the ligand, but with Cap-R5-NH2, over 95% of the DNA partitioned into the organic phase. This observation is consistent with the decreased accessibility of water to electron-deficient guanine species located in DNA bound with a C-8 derivative of spermine.14 Atomic Force Microscopy. Particle sizes and the interaction of the neutral detergent Pluronic F-127 with mixtures of Cap-R5NH2 and plasmid DNA were examined with atomic force microscopy. Figure 7A shows the typical appearance of a plasmid treated with the ligand Cap-R5-NH2 in the presence of Pluronic F-127. Many objects with diameters in the micrometer size range are visible, which is consistent with the DLS and NTA data. We note, however, that the AFM image was made with a dried sample, and may therefore poorly reflect particle sizes in an aqueous environment. There are relatively few objects with sizes expected of regular Pluronic micelles, which are significantly smaller circular particles with sizes less than 100 nm (Figure 7B).49 Nor are individual plasmids visible, which would be filamentous objects several hundred nanometers in length.15 This image suggests that the majority of the plasmid is aggregated and that the detergent is associated with these aggregates.

’ CONCLUSIONS In conclusion, we have shown that the conjugate formed when the C-8 fatty acid caprylic acid is linked to the N-terminus of the cationic oligopeptide penta-arginine is able to condense plasmid DNA. The resulting condensate forms under conditions very similar to those required for penta-arginine (R5) itself. Both confer an extensive protection against the DNA damage produced by the indirect effect of ionizing irradiation (mediated by 1736

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Biomacromolecules water derived radical species). However, the condensate produced using Cap-R5-NH2 is far more lipophilic than that formed with R5. Because the synthesis of peptide derivatives is a simple process, the conditions we describe here offer a convenient experimental system with which to further restrict the access of water to DNA14 and to examine the interaction of condensed plasmid DNA with lipids or lipophilic materials. Examples would be the interaction with DNA of radio-sensitizing or radioprotective drugs that partition poorly into aqueous solutions or the association with a neutral surfactant.49

’ ASSOCIATED CONTENT

bS

Supporting Information. UV
vis spectra from 200 to 800 nm of pUC18 plasmid DNA as a function of the concentration of the ligand Cap-R5-NH2. Hydrodynamic radius distributions of pUC18 plasmid DNA in the absence and presence of Cap-R5-NH2 and or Pluronic F-127, as determined by DLS. This material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

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