Tamon Kusumoto

Michel Fromm
email: michel.fromm@univ-fcomte.fr

Pierre Cloutier

Andrew D Bass

Léon Sanche

Rémi Barillon

Tomoya Yamauchi

Elucidation of the Two-step Damage Formation Process of Latent Tracks in Poly(allyl diglycol carbonate), PADC: Role of Secondary Low-energy Electrons

The aim of the present study is to evaluate the mechanisms by which secondary electrons of low energy degrade poly(allyl diglycol carbonate) (PADC) when it is used as a Solid State Nuclear Track Detector. We measured by time of flight mass spectrometry, the positive and negative ions desorbed by the impact of 5-30 eV electrons on three monolayer thick films of various PADC surrogate molecules, which contained ether and/or carbonyl (C=O) moieties. As cleavages within the molecules progress, most anion yields are seen to decrease with an increasing cumulative number of incident electrons (i.e., fluence). In contrast, the yield of O -at incident energies > 15 eV, which is associated with damage at a carbonyl group, increases monotonically with fluence. This finding implies that cleavage of carbonyl groups occurs after damage to other oxygen containing groups within the surrogates. The present result corroborates the previously postulated two-step degradation process in PADC irradiated with high-energy electrons and 222 nm UV photons. More generally, these results underline the importance of considering the role of low energy secondary electrons in the deterioration of high-sensitivity energetic particle detectors.

Table 1 List of fragments desorbed from condensed FDA, DC and DGMEA. Poly(allyl diglycol carbonate), PADC, often called CR-39 TM (its trade mark "Columbia Resin #39") is considered as the most sensitive Solid State Nuclear Track Detector, SSNTD. [START_REF] Cartwright | A nuclear-track-recording polymer of unique sensitivity and resolution[END_REF], [START_REF] Fleischer | Nuclear Tracks in Solids: Principles and Applications[END_REF] Unlike other SSNTDs, PADC can detect energetic protons (and neutron recoil nuclei) as well as heavy ions, in mixed radiation fields without background noise. Due to this advantage, it has been utilized in diverse applications, such as space radiation dosimetry (3)- [START_REF] Kodaira | Control of the detection threshold of CR-39 PNTD for measuring ultra heavy nuclei in galactic cosmic rays[END_REF] and laser driven ion acceleration experiments. [START_REF] Fukuda | Energy Increase in Multi-MeV Ion Acceleration in the Interaction of a Short Pulse Laser with a Cluster-Gas Target[END_REF], [START_REF] Kanasaki | The precise energy spectra measurement of laser-accelerated MeV/n-class high-Z ions and protons using CR-39 detectors[END_REF] At least six books have been dedicated to SSNTD applications. ( 8)- [START_REF] Zhou | CR-39 Plastic Nuclear Track Detectors in Physics Research[END_REF] Beginning in the 1990s, molecular damage (i.e., loss/generation of chemical functions) within PADC has been examined using FT-IR spectrometric methods for a wide range of stopping powers, specifically from 1.2 to 12,000 eV/nm, ( 14)- [START_REF] Mori | Radiation chemical yields for loss of ether and carbonate ester bonds in PADC films exposed to proton and heavy ion beams[END_REF] with a view to developing new SSNTD materials with desired track registration sensitivity from the viewpoint of molecular re-arrangement. Figure 1 (a) represents a repeat unit of PADC, which contains the radiation-sensitive moieties of an ether in the center and two carbonate esters in symmetrical positions. Such radiation-sensitive moieties are connected by "polyethylene-like" networks formed during the polymerization, which are considered as the radiation-tolerant moiety of the polymer. [START_REF] O'donnell | Radiation Chemistry of Polymers. The Effects of Radiation on High-Technology Polymers[END_REF] From a complete analysis of the existing available data, ( 14)- [START_REF] Mori | Radiation chemical yields for loss of ether and carbonate ester bonds in PADC films exposed to proton and heavy ion beams[END_REF] a two-step damage formation model of the latent track in PADC was deduced. This mechanism supposes that the carbonate ester can only be lost on the ester in the same PADC unit. [START_REF] Kusumoto | Sudden increase of the radiation chemical yield for loss of carbonate ester in PADC detector where the track overlapping of 28 MeV electrons becomes significant[END_REF] Based on the conventional track-overlapping model, which expresses the distribution of ion tracks using random numbers, we clarified that at least two electron hits are required to break the carbonate ester in PADC. [START_REF] Kusumoto | Sudden increase of the radiation chemical yield for loss of carbonate ester in PADC detector where the track overlapping of 28 MeV electrons becomes significant[END_REF] Indeed, it was quantitatively observed that the damage to PADC irradiated with protons and heavy ions follows a mechanism in which the amount of ether lost is always greater than that of carbonate ester and other functional groups. To further investigate this hypothesis and determine the mechanisms of PADC degradation, we measured the damage induced by low-energy electrons (LEEs, 5 -30 eV) to surrogates of PADC. Since LEEs are produced in large numbers in organic media (~340,000/MeV of deposited primary energy), [START_REF] Pimblott | Production of low-energy electrons by ionizing radiation[END_REF] we expect their interactions and the damage they induce in PADC to be significant and related to the mechanisms of degradation. In fact, the large amount of LEEs produced around the track of energetic protons in PADC has recently been determined by computer simulations. [START_REF] Fromm | Low energy electrons and swift ion track structure in PADC[END_REF] In the present study, we investigated damage to hydrocarbon molecules condensed as thin films on a metal substrate in ultra-high vacuum (UHV). The measurements were performed by measuring desorption of negative and positive ions, induced by electrons impinging on the films with energies between 5 and 30 eV. Since neither PADC nor diethyleneglycol bis allyl carbonate, (i.e. the monomer in PADC) are sufficiently volatile to be introduced as vapors into the apparatus, the electron stimulated desorption (ESD) currents were recorded with films made of condensed surrogates of PADC. We also note that previous anion ESD experiments have shown that LEEs induce C-O and C-H bond cleavages in small ethers and hydrocarbon molecules. [START_REF] Bogdan | A dramatic difference between the electron-driven dissociation of alcohols and ethers and its relation to Rydberg states[END_REF], [START_REF] Rowntree | Anion yields produced by low-energy electron impact on condensed hydrocarbon films[END_REF] 2. Experimental section

Materials

The surrogates of PADC, whose nomenclatures are presented in Fig. 1 (b),(c) and (d), are formaldehyde diethyl acetal (FDA), diethyl carbonate (DC) and diethylene glycol monoethyl ether acetate (DGMEA), all of which were obtained from SIGMA-ALDRICH Co. LLC. Two ethers, methylene and ethyl groups compose FDA.

In contrast, DC contains only a carbonate ester groups in its center. DGMEA contains both radio-sensitive moieties of ether and carbonate groups which are separated by ethyl groups.

LEE irradiation and TOF mass spectrometry

For each target, three monolayers of a particular surrogate molecule were condensed into three monolayers on a platinum substrate held at 50 K in a stainless steel UHV chamber pumped to 2×10 -10 Torr installed in Sherbrooke University, Canada. [START_REF] Hedhili | Electron stimulated desorption of anionic fragments from films of pure and electron-irradiated thiophene[END_REF], [START_REF] Bazin | Electron induced dissociation in condensed-phase nitromethane I: desorption of ionic fragments[END_REF] LEE irradiations were performed with a pulsed electron beam (Current: 1.1 nA, Beam size: 3±0.5 mm 2 ) by a Kimball Physics ELG-2 electron gun. Electrons impinged onto the molecules at an incident angle of 45° from the substrate normal. The desorbed ions yields were measured as functions of incident electron energy (ion yield functions) by recording mass spectra for 10 5 electron pulses (equivalent to 1.4×10 11 electrons) at incrementally increasing incident electron energies with steps of 0.25 eV. To minimize the effects of sample charging, yield functions were recorded for 5 -15 eV on one film, and then, for 15 -30 eV on another. Desorbed fragments were injected into a time of flight (TOF) mass spectrometer (Kore Technology R-500) to record the yields and mass to charge ratios (m/z) of desorbed anions and cations. [START_REF] Bazin | Electron induced dissociation in condensed-phase nitromethane I: desorption of ionic fragments[END_REF]

Results and discussion

In the present ESD experiments, desorption of anions essentially stems from two distinct processes: dissociative electron attachment (DEA) and dipolar dissociation (DD). DEA to a molecular target AB can be represented as, [START_REF] Bass | Charge particle ant photon interactions with matter: Chemical, physicochemical and biological consequences with applications[END_REF] 𝐴𝐵 + 𝑒 → 𝐴𝐵 * → 𝐴 + 𝐵 (1) where AB *-is the intermediate transient anion, which dissociates into a stable anion A - and a neutral species B within a short lifetime of the order of a vibrational period of AB *-. DEA is a resonant scattering process due to involvement of the transient (compound) anion state formed by both the molecule and incident electron. The DD process corresponds to the following reaction scheme, [START_REF] Bass | Charge particle ant photon interactions with matter: Chemical, physicochemical and biological consequences with applications[END_REF] 𝐴𝐵 + 𝑒 → 𝐴𝐵 * + 𝑒 → 𝐴 + 𝐵 + 𝑒

In DD, the electronically excited molecule fragments into an ion pair of A -and B + .

The cross section for the DD process is usually non-resonant and hence increases monotonically with electron energy; i.e., in our data, from threshold (12 -15 eV) to maximum values at higher energies between ~50 and ~100 eV. [START_REF] Bass | Charge particle ant photon interactions with matter: Chemical, physicochemical and biological consequences with applications[END_REF] The desorption of cationic fragments also proceeds by dissociative ionization (DI) via excitation of dissociative molecular cationic states of the molecule, i.e.,

AB + e -→ A + B + + 2e -

Threshold energies for the DI process will be higher than for DD by at least the electron affinity of the electronegative fragment A; maximum yields are observed at similar energies to DD processes. DD and DI processes by electronic excitation are examples of non-resonant scattering processes.

Desorbed negative and positive fragments are listed in Table 1. Some examples of anion and cation yield functions are illustrated in Fig. 2 and in Fig. 3, respectively. Circle, square and diamond symbols indicate, respectively, the anion and cation yields from FDA, DC and DGMEA. The yields of H -(m/z = 1) are at least three orders of magnitude higher than all other anions, consistent with previous results based on condensed hydrocarbon targets. (21)(21) Yields of CO -or CO2 -(m/z = 28 or 44), whose neutral forms are known to originate from the segmentation of carbonate ester groups [START_REF] Yamauchi | Yields of CO2 formation and scissions at ether bonds along nuclear tracks in CR-39[END_REF] were not observed for any target molecule [START_REF] Lepage | Low-energy electron scattering cross section for the production of CO within condensed acetone[END_REF], [START_REF] Orzol | Reactions in nanofilms of trifluoroacetic acid (CF3COOH) driven by low energy electrons[END_REF] , due to the negative electron affinities of CO and CO2 ground states. [START_REF] Orzol | Reactions in nanofilms of trifluoroacetic acid (CF3COOH) driven by low energy electrons[END_REF] Since the detection probabilities of the ions vary with their mass, momentum and their interactions in the film prior to desorption, [START_REF] Hedhili | Electron stimulated desorption of anionic fragments from films of pure and electron-irradiated thiophene[END_REF], [START_REF] Bazin | Electron induced dissociation in condensed-phase nitromethane I: desorption of ionic fragments[END_REF] it is not possible to infer relative cross sections for ion production from the measured ion yield. The anion yields of CH3 -(m/z = 15) are represented as a function of the incident electron energy in Fig. 2 (b). Most likely, the CH3 -originates from cleavages at the positions labeled "1" in Fig. 1. CH -and CH2 -(m/z = 13 or 14) are also observed and may originate from proton transfer during the same DEA process that generates the CH3 moiety. In Fig. 2 (b), we observe DEA resonances at 9.0 eV, that can be assigned to the decay of core-excited transient anions. [START_REF] Bass | Charge particle ant photon interactions with matter: Chemical, physicochemical and biological consequences with applications[END_REF] Above 15 eV, the recorded monotonic increase of the yields corresponds to DD. Similar trends are observed in the anion yield functions of H -, C2H4 -, and C2H5O -(m/z = 1, 28, and 45).

In contrast to the high anion yields of H -, the cation signal of H + is barely observable. No noticeable structures are seen in the cation yield function of CH3 + (Fig. 3 (a)), whose amplitude rises significantly above 18 eV. The behavior of CH3 + yield function is almost identical to those of all other cations, as depicted in Fig. 3 Qualitatively speaking, desorption of OH -are fairly low compared to those of other anions. The desorption of OH -from these molecules suggests that some rearrangement or "scrambling" during dissociation is possible, for example to allow proton transfer from a carbon atom to a departing O -ion. The formation of OH -by the reactive scattering of O -in hydrocarbon containing films has also been reported. [START_REF] Kühn | Formation and Dissociation of Negative-Ion Resonances in Methanol and Allylacohol[END_REF] In such cases nevertheless, OH -yield was significantly smaller (i.e., < 10%) that of the O - signal.

In previous experiments with UV photons at a wavelength of 222 nm, decarboxylation accompanied by the release of CO and CO2 gas was observed by IR spectroscopy along with the loss of carbonyl signal in PADC. [START_REF] Sakamoto | Quantum yields for loss of carbonate ester bonds in polymeric nuclear track detectors under 222 nm UV radiation[END_REF] It was additionally found that carbonyl groups were not cleaved until neighboring ether or C-O-C, which composes carbonate ester, were damaged. In the present study, due to the variety of fragment desorbed, especially at masses ≥ 25 amu, it seems likely that carbonyl moieties are once again only damaged after cleavages of the ether or ester in the same molecule has occurred.

As mentioned above, the desorption yields of O -and OH -from DGMEA are barely detected for electron energies below 15 eV. Thus, we can say that desorption of O -is due to DD at the carbonyl. To test that hypothesis, we investigated the variation of anion yields with electron exposure to at two particular energies. Figure 4 and5 show how the yields of CH3 -, O -and C2H3O2 -desorbed from DGMEA vary during extended electron irradiation at incident electrons with energies of 9 and 30 eV, respectively (i.e.

below and above the DD threshold). In the figures, the yields are normalized to their initial values, obtained after exposure to 1.4×10 11 electrons. When samples are exposed to electrons at 9 eV, the anion yields of CH3 -, O -and C2H3O2 -are observed to decrease slightly during prolonged irradiation (Figure 4). When samples are exposed to electrons at 9 eV, the anion yields of CH3 -, O -and C2H3O2 -are observed to decrease slightly during prolonged irradiation (Figure 4). This small decrease in signal likely reflects the gradual loss of intact DGMEA molecules from the film (and more specifically near the film/vacuum interface, where most desorbed ions originate). Much more pronounced decreases in desorption signal are reported in Fig. 5 for CH3 -and C2H3O2 -fragments from films under electron irradiation at 30 eV (Figure 5). Once again, this decrease is plausibly related to the depletion within the film of intact molecules of fragments capable of dissociating to produce these anions. However, at 30 eV, net positive sample charging is possible due to ionization occurring within the film, secondary electron emission, and the trapping of cations formed by DI and DD. Such charging would act to hamper the desorption of low kinetic energy anions. However, similar behaviors under electron bombardment of the normalized CH3 -and C2H3O2 -desorption signals (Figure 5) suggest that the depletion of intact DGMEA molecules must be the favored explanation for the observed decrease with fluence. These signals surely derive from close to the ends of the DGMEA molecule, by scission of bonds 1 (or 11) and 4 (see Figure 1 (d)). The kinetic energies of these fragment ions are likely different; if film charging were the cause of their variation with fluence, then it would me most surprising for them to behave so similarly.

In contrast to the other signals reported in Fig. 5, the relative yield of O -rises monotonically with increasing exposure to 30 eV electrons. This specific behavior indicates that some electron-induced modification of molecules in the sample produces new species in the film, for which the probability of O -desorption at 30 eV is sufficiently greater than for intact DGMEA. Such a behavior demonstrates that cleavages of carbonyl are promoted by longer exposure times (i.e. by increasing the probability that an electron hit has already occurred elsewhere in the molecules). In addition, we can deduce from this analysis that at least two electrons are required to damage the carbonyl group in DGMEA. Following initial electron-induced damage, heavy fragment species remaining in the film are subsequently excited by other incident electrons, leading to carbonyl breakage in DGMEA. Therefore, it is highly probable that the ester in DGMEA is cleaved after damage to the ether. The present result confirms the previously suggested two-step damage formation model deduced from FT-IR measurements. [START_REF] Kusumoto | Sudden increase of the radiation chemical yield for loss of carbonate ester in PADC detector where the track overlapping of 28 MeV electrons becomes significant[END_REF] This strongly corroborates the concept that the number of LEEs, which hit a molecule, may represent an appropriate parameter to describe the damage formation process of PADC and more generally in polymers.

Conclusion

In For a more detailed understanding of the mechanism of latent track formation in PADC, it may be possible to use the DD related O -desorption signal from DGMEA at 30 eV, and so probe such processes at electron energies below the threshold for anion desorption at ~ 5 eV, for example via low lying shape resonances. A similar approach has been used to monitor damage in CF2Cl2 and CH3COH. [START_REF] Sakamoto | Quantum yields for loss of carbonate ester bonds in polymeric nuclear track detectors under 222 nm UV radiation[END_REF] Alternatively, changes in high-resolution-electron-energy-loss vibrational or electronic spectra may provide useful information. [START_REF] Sakamoto | Quantum yields for loss of carbonate ester bonds in polymeric nuclear track detectors under 222 nm UV radiation[END_REF] Finally, we note that the radical and ions created by the LEEs may undergo subsequent chemical reactions (e.g. in the case of water radiolysis see Marignier et al.). [START_REF] Sakamoto | Quantum yields for loss of carbonate ester bonds in polymeric nuclear track detectors under 222 nm UV radiation[END_REF] Fig. 1.

Figure 1

 1 Figure 1 Chemical structures of (a) a repeat unit of PADC; and molecular structures of (b) formaldehyde diethyl acetal, FDA, (c) diethyl carbonate, DC, and (d) diethylene glycol monoethyl ether acetate, DGMEA.

Figure 2

 2 Figure 2 The anion yields of (a) H -, (b) CH3 -, (c) O -, (d) OH -, (e) C2H4 -, and (f) C2H5O - as a function of the incident electron energy. Yields from FDA, DC and DGMEA are indicated respectively with circle, square and diamond symbols.

Figure 3

 3 Figure 3 The cation yields of (a) CH3 + , (b) C2H3 + , (c) C2H3O + and (d) C2H3O2 + as a function of the incident electron energy. The yields from FDA, DC and DGMEA are represented as circle, square and diamond symbols, respectively.

Figure 4

 4 Figure 4 The relative anion yield of CH3 -(open circles), O -(open triangles), C2H5O2 - (open squares) from DGMEA, as a function of the number of electrons. Full lines in the graph are guides for the eye.

Figure 5

 5 Figure 5 The relative anion yield of CH3 -(open circles), O -(open triangles), C2H5O2 - (open squares) from DGMEA, as a function of the number of incident 30 eV electrons.Full lines in the graph are guides for the eye.

  (b), (c) and (d). Judging from these trends, it can be concluded that the threshold of DD lies between 15 and 18 eV. We also observe a clear peak in the anion yield function of O - (m/z = 16) from DC at 9.5 eV (Fig.2 (c)). On the other hand, the O -yield functions from FDA and DGMEA do not exhibit noticeable resonances. Moreover, the O -yield from FDA does not rise significantly above 16 eV. Thus while O -anions desorption from ethers in FDA is barely detected, the release fragments with m/z = 25, 26, 27, 28, 29, 41, 43 and 45 from FDA reveals significant ether damage. In contrast, we observe a drastic increase of the O -yield from DGMEA above ~15 eV incident electron energy.Therefore, O -desorption from DGMEA arises from electronic excitation of the double bond in location labeled "2", which has no analog in FDA. The yield function of OH - (m/z=17) is also seen in Fig.2 (d). In the OH -yield function, the resonances are observed around 10 eV and a monotonic rise in yield is confirmed above 15 eV.

  the present study, we investigated the role played by low energy secondary electrons, in the creation of latent tracks in PADC. For this purpose, we measured by TOF mass spectrometry the electron stimulated desorption of anions and cations from multilayer films of three different PADC surrogates FDA, DC and DGMEA. With the exception of O -desorption from FDA and DGMEA, the anion yield functions from these films exhibited resonance near 9 eV. That could be assigned to DEA. In contrast, the only O -yield function showing a resonance at this energy arises from DC, i.e., no resonance feature is apparent in measurements for FDA and DGMEA. The anion yields of O -from DC and DGMEA increase monotonically with incident electron energies above 15 eV, where molecular cleavages are governed mainly by DD. The O -yield from FDA, which does not contain the carbonyl group, was very low and almost constant over the entire energy range. We therefore surmise that desorption of O -from DGMEA above 15 eV is essentially due to DD at the carbonyl. Additionally, we measured the variation of fluence, at 9.0 and 30 eV, i.e., at electron impact energies lying respectively below and above the energetic threshold for DD. When DGMEA was irradiated with 9 eV electrons, the anion yields of CH3 -, O -and C2H3O2 -decreased with the number of incident electrons. As the number of 30 eV electrons increased, the relative anion yields of CH3 -and C2H3O2 -also decreased. In contrast, the yield of O - increased significantly with fluence. This behavior supports the previously proposed degradation mechanism postulated from the results obtained by FT-IR spectrometry of PADC irradiated with 28 MeV electrons and 222 nm UV photons. In fact, the ensemble of our results confirm the previously suggested two-step damage formation model deduced from FT-IR measurements. They validate the concept that the number of LEEs, which hit a molecule, may represent an appropriate parameter to describe the details of damage formation in a latent track of PADC, and more generally of polymers, when such compounds are exposed to high-energy radiations.

Fig. 2 .Fig

 2 Fig. 2.

Acknowledgements

This study was supported by the JSPS KAKENHI Grant Number 16K05002, the Canadian Institutes of Health Research (MOP81356) and the funds of Kobe University for the cotutelle program with the Université de Strasbourg.


T T Khalil

O Boulanouar

O Heintz

M Fromm

Auto-assembly of nanometer thick, water soluble layers of plasmid DNA complexed with diamines and basic amino acids on graphite: greatest DNA protection is obtained with arginine

Keywords: DNA thin film, diamines, amino acids, plasmid relaxation

We have investigated the ability of diamines as well as basic amino acids to condense DNA onto highly ordered pyrolytic graphite with minimum damage after re-dissolution in water. Based on a bibliographic survey we briefly summarize DNA binding properties with diamines as compared to basic amino acids. Thus, solutions of DNA complexed with these linkers were drop-cast in order to deposit ultra-thin layers on the surface of HOPG in the absence or presence of Tris buffer. Atomic Force Microscopy analyses showed that, at a fixed ligand-DNA mixing ratio of 16, the mean thickness of the layers can be statistically predicted to lie in the range 0-50 nm with a maximum standard deviation ± 6 nm, using a simple linear law depending on the DNA concentration. The morphology of the layers appears to be liganddependent. While the layers containing diamines present holes, those formed in the presence of basic amino acids, except for lysine, are much more compact and dense. X-ray Photoelectron Spectroscopy measurements provide compositional information indicating that, compared to the maximum number of DNA sites to which the ligands may bind, the basic amino acids Arg and His are present in large excess. Conservation of the supercoiled topology of the DNA plasmids was studied after recovery of the complex layers in water. Remarkably, arginine has the best protection capabilities whether Tris was present or not in the initial solution.

Introduction

Nanometer-scale deposits of plasmid DNA (closed circular DNA double strands) which can be dissolved in water have shown considerable interest in radiobiological experiments aiming at

A C C E P T E D M A N U S C R I P T

deepening and refining understanding of the fundamental principles of how low-energy electrons, ions or VUV, X-rays and -rays interact with DNA [START_REF] Boudaiffa | Resonant formation of DNA strand breaks by low energy (3-20 eV) electrons[END_REF][START_REF] Brun | Damage induced to DNA by lowenergy (0-30 eV) electrons under vacuum and atmospheric conditions[END_REF][START_REF] Alizadeh | The Role of Humidity and Oxygen Level on Damage to DNA Induced by Soft X-rays and Low-Energy Electrons[END_REF][START_REF] Lacombe | DNA strand breaks induced by low keV energy heavy ions[END_REF][START_REF] Śmiałek | Preparation of DNA films for studies under vacuum conditions The influence of cations in buffer solutions[END_REF][START_REF] Śmiałek | The influence of the substrate temperature on the preparation of DNA films for studies under vacuum conditions[END_REF][START_REF] Folkard | Critical energies for ssb and dsb induction in plasmid DNA by vacuum-UV photons: an arrangement for irradiating dry or hydrated DNA with monochromatic photons[END_REF]. In such experimental approaches it is vital to keep DNA plasmids as undamaged as possible (i.e. in their supercoiled topology) before treatment by radiation. Actually, when performing the plasmid relaxation assay, the percentages of supercoiled, open circular and linear forms were quantified; the two latter being the signatures of DNA single and double strand breaks respectively. It should be noted that in most studies which aimed at studying radiation effects at the molecular level, plasmid DNA thin films were used under the form of freezedried deposits. Sometimes however, irradiation arrangement was modified to allow humidified DNA [START_REF] Brun | Damage induced to DNA by lowenergy (0-30 eV) electrons under vacuum and atmospheric conditions[END_REF][START_REF] Alizadeh | The Role of Humidity and Oxygen Level on Damage to DNA Induced by Soft X-rays and Low-Energy Electrons[END_REF][START_REF] Lacombe | DNA strand breaks induced by low keV energy heavy ions[END_REF] to be exposed to radiation under atmospheric conditions. Lyophilized plasmid DNA layers unfortunately present the disadvantage of an uneven film surface coverage and thickness [START_REF] Śmiałek | Preparation of DNA films for studies under vacuum conditions The influence of cations in buffer solutions[END_REF]. In addition, with the freeze-dried layers, it is impossible to perform experiments under vacuum with so-called "pure DNA", namely with no stabilizers added to the sample before the evacuation process. Re-dissolution of the DNA plasmids in water is a sine qua non condition if a plasmid relaxation assay is to be subsequently performed. Experimentally, the presence of a chemical buffer to stabilize the plasmid DNA topology therefore appears to be a key requirement [START_REF] Śmiałek | The influence of the substrate temperature on the preparation of DNA films for studies under vacuum conditions[END_REF]. Besides, under biological conditions, nuclear DNA does not occur in free linear strands; quite the opposite, it is highly condensed and wrapped around histones [START_REF] Luscombe | An overview of the structures of protein-DNA complexes[END_REF]. Recently, we described a way to produce nanometer-scale DNA films of uniform and directly measurable thicknesses on conductive substrates of highly ordered pyrolytic graphite (HOPG) [START_REF] Boulanouar | Soft adsorption of densely packed layers of DNA-plasmid • 1,3-diaminopropane complexes onto highly oriented pyrolitic graphite designed to erode in water[END_REF]. The technique employed the aliphatic diamine, 1,3-diaminopropane in its doubly protonated form (Dap 2+ ) at neutral pH, to bind plasmid DNA molecules to each other. Diamines and polyamines are indeed actively involved in a considerable number of metabolic events, including DNA replication, protein synthesis, and cell growth [START_REF] Sakai | Effects of polyamines on the structure and reactivity of tRNA[END_REF][START_REF] Tabor | 1,4-Diaminobutane (putrescine), spermidine, and spermine[END_REF]. Plasmid DNA films structured with Dap 2+ as linkers have proven excellent capabilities under low energy electron (0-20 eV) impact with the possibility, for the first time, to measure the absolute cross-section [START_REF] Boulanouar | Absolute cross section for loss of supercoiled topology induced by 10 eV electrons in highly uniform /DNA/1,3diaminopropane films deposited on highly ordered pyrolitic graphite[END_REF] for the loss of plasmid DNA supercoiled topology at the resonance energy of 10 eV due to dissociative electron attachment (DEA). This was achieved thanks to the uniformity of the layers at the nanometer scale. Moreover, electron stimulated desorption (ESD) analyses [START_REF] Boulanouar | Dissociative electron attachment to DNA-diamine thin films: impact of the DNA close environment on the OH-and O-decay channels[END_REF] of such films have revealed that the presence of the Dap 2+ molecule in the close vicinity of the phosphate DNA moieties has a profound impact on the ESD yields of anions with notably a quasi-entire

A C C E P T E D M A N U S C R I P T

suppression of the O -desorption channel (generally observed when using "pure" or lyophilized DNA) in favor of the appearance of a high OH -desorption yield. This indicates that the close environment of the DNA macromolecule in more realistic biological conditions should specifically affect the way in which it responds to radiation injury. It thus appears particularly relevant to search for specific (i.e. biogenic) DNA ligands that would make it possible to produce nanometer-scaled plasmid DNA layers with the property of being soluble in water with the only severe damage due to radiation effects.

In order to tightly pack the large DNA macro-anions into the nucleus of a biological cell, life has selected to use molecular tails bearing in particular protonated amino moieties (i.e.

amino acids positively charged at neutral pH). In eukaryotic cell chromosomes, the basic amino acids arginine, lysine and histidine play a crucial role in packing DNA into chromatin [14,[START_REF] Teif | Condensed DNA: condensing the concepts[END_REF]. It should be underlined that in addition to basic amino acids, polyamines are also involved in the mechanism of DNA folding-unfolding in biological cells [START_REF] Manning | The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides[END_REF] as well as stabilization of the negative charges of DNA or RNA [START_REF] Koculi | Counterion charge density determines the position and plasticity of RNA folding transition states[END_REF].

In vitro condensation of DNA has been studied both theoretically [START_REF] Bloomfield | DNA condensation by multivalent cations[END_REF][START_REF] Manning | The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides[END_REF] and experimentally [START_REF] Koltover | DNA condensation in two dimensions[END_REF] and still remains of topical interest, especially when surface-mediated delivery of DNA is foreseen [START_REF] Storrie | Sustained delivery of plasmid DNA from polymeric scaffolds for tissue engineering[END_REF][START_REF] Jewell | Surface-mediated delivery of DNA: Cationic polymers take charge[END_REF][START_REF] Hujaya | Multilayered thin films from poly(amido amine)s and DNA[END_REF]. To condense DNA in aqueous media, at least the presence of a salt is required to neutralize DNA charges and decrease DNA-DNA poly-anion repulsion. A more sophisticated and efficient way to condense DNA consists of inducing attractive interactions between the DNA macromolecules by using multivalent cationic charged ligands (multivalent metal ions, inorganic cations, polyamines, protamines, peptides, lipids, liposomes and proteins) [START_REF] Teif | Condensed DNA: condensing the concepts[END_REF].

Using the soft adsorption (drop-casting) protocol developed for fabricating Dap-DNA layers deposited on HOPG [START_REF] Boulanouar | Absolute cross section for loss of supercoiled topology induced by 10 eV electrons in highly uniform /DNA/1,3diaminopropane films deposited on highly ordered pyrolitic graphite[END_REF], we studied the ability of aliphatic diamines of ethylene diamine (Eda), 1,4-diaminobutane (putrescine or Put), 1,5-diaminopentane (cadaverine or Cad) as well as basic amino acids histidine (His), arginine (Arg) and lysine (Lys) to condense plasmid DNA into dense layers on the surface of HOPG in the presence but also in the absence of the Tris-EDTA buffer. Atomic Force Microscopy (AFM) will be used to check and characterize the deposits formed on freshly cleaved HOPG with a defined mixing ratio R (R = [Ligand] / [DNAphosphate]). The relationship thickness (in nanometers) of the layer versus C DNA at a fixed mixing ratio will be established for Dap and for Arg which gave the best deposit qualities (the cases of His, Lys, Eda, Put, Cad nevertheless are also discussed). Special attention will be paid

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to the integrity of the initial proportion of supercoiled topology of the plasmids over all the different experimental steps using gel electrophoresis measurements, including the last step that consists in recovering the plasmids in water for further analysis. The proportion of ligands (diamines and amino acids) remaining in the layers was then determined by means of X-ray Photoelectron Spectroscopy (XPS). The relationship between the type of ligand binding site and the stability of the complexes, especially maintenance of the supercoiled topology of the DNA plasmids is discussed.

Materials and methods

Chemicals and Materials

Preparation of [DNA • ligand] complexes

Ethylene diamine dihydrochloride (≥98%), 1,3-diaminopropane dihydrochloride (≥98%),

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Layer Preparation and AFM investigation

The DNA-Ligand deposits were prepared by soft adsorption: A droplet of 50 μl of the [DNA • Ligand] mixture was deposited onto the surface of freshly cleaved graphite (HOPG, ZYA grade, NT-MDT, NanoAndMore, France) and incubated for 15 min. Next, the surplus solution was removed using filter paper. This protocol ensures that a circular smear of constant diameter 8 ± 1 mm is created on the freshly cleaved HOPG surface (it can be observed by the naked eye). Then, the surface was dried in clean air for 3 min performed.

AFM images were made using a Molecular Imaging scanning probe microscope (Agilent, USA). The topography of the surface was recorded in air, at standard temperature and pressure, with silicon nitride tips coated with aluminum (NanoAndMore, France) at a resonant frequency of 300 kHz in the tapping mode. The way the thickness measurements were performed at the nanometer scale was presented in detail elsewhere [START_REF] Boulanouar | Soft adsorption of densely packed layers of DNA-plasmid • 1,3-diaminopropane complexes onto highly oriented pyrolitic graphite designed to erode in water[END_REF][START_REF] Boulanouar | Dissociative electron attachment to DNA-diamine thin films: impact of the DNA close environment on the OH-and O-decay channels[END_REF]. Let us briefly recall here that, unlike the situation where the tapping mode is used to observe the layers, when working in the contact mode, the layers can be swept away leaving a tear over the entire scanned field revealing the naked HOPG surface (see Fig. 4, inset). An additional scan in the tapping mode over a greater field makes the thickness of the layer accessible to AFM measurement.

Instruments and Methods

Gel electrophoresis measurements were based on 1% agarose in TAE buffer at 6.7 V.cm -1 for 7 minutes and 5.0 V.cm

Results

Before going into details with the experimental part of this work, we would like to briefly describe the main features of the linkers used in the present study (especially their chemical structure and properties) and identify the known DNA binding sites of those linkers, including Tris buffer, as exactly as possible. A brief bibliographic survey is a prerequisite for a better understanding of the differences observed, notably in terms of plasmid DNA protection all along the different steps with regard to maintaining the supercoiled topology.

Ligands

The molecules used to study plasmid DNA condensation and deposition properties are presented in Fig. 1.

Fig. 1. Linear diamines: ethylene diamine (Eda), 1,4-diaminobutane (putrescine or Put), 1,5-diaminopentane (cadaverine or Cad) as well as basic amino acids histidine (His), arginine (Arg) and lysine (Lys) Protonated amino groups which contribute to a bulk positive charge of the molecules at neutral pH are in bold blue. Due to triple resonance stabilization, the guanidinium group of the arginine (Arg) amino acid presents 3 potentially positively-charged nitrogen atoms [START_REF] Schug | Noncovalent binding between Guanidinium and anionic Groups: focus on biological-and synthetic-based Arginine/Guanidinium interactions with phosph[on]ate and sulf[on]ate residues[END_REF].

In the aqueous environment, diamines such than Eda, Dap, Put and Cad have pKa 1 and pKa 2 values greater than 7, except Eda, whose pKa 2 was measured in the range 6.8 -7.5 by others [START_REF] Perrin | Dissociation Constants of Organic Bases in Aqueous Solution[END_REF][START_REF] Perrin | Dissociation Constants of Organic Bases in Aqueous Solution[END_REF]. Recently refined pKa calculations [START_REF] Bryantsev | pKa calculations of aliphatic amines, diamines, and aminoamides via density functional theory with a Poisson-Boltzmann continuum solvent model[END_REF] confirm the quoted experimental data. As for the three basic amino acids, their isoelectric point (pI) is always greater than 7 [START_REF] Lehninger | Lehninger Principles of Biochemistry[END_REF], which implies that in aqueous media at pH=7, all basic amino acids are ionized (zwitterionic form at neutral pH). On the other hand, only arginine and lysine side-chains are ionized at neutral pH, for the histidine side-chain, pKa=6 [START_REF] Schug | Noncovalent binding between Guanidinium and anionic Groups: focus on biological-and synthetic-based Arginine/Guanidinium interactions with phosph[on]ate and sulf[on]ate residues[END_REF]. The latter are shown in blue in Fig. 1 for clarity.

To ensure ionization of the diamines and side-chain amino groups in water, the diamines used were purchased under the form of hydrochloride salts. We may thus consider that all

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the ligands used are mostly in their doubly-ionized state for diamines with an ionized sidechain for amino acids, at neutral pH in water. Tris buffer (pKa = 8.30), which contains itself an amino group, is protonated at neutral pH. EDTA is also present in the chemical buffer solution but in a 10-fold lesser proportion, it is negatively charged.

3.2. Ligand binding sites.

Diamines

In this study, diamines Eda, Dap, Put and Cad all are considered as doubly-protonated in aqueous solution. As mentioned by Korolev and co-workers [START_REF] Korolev | Molecular dynamics simulation study of oriented DNA with polyamine and sodium counterions: diffusion and averaged binding of water and cations[END_REF], diamines form only shortlived contacts with the electro-negative binding sites of DNA and do not form any structurally defined complexes. Using molecular dynamics (MD) simulations these authors have shown that diamines (Dap, Put and Cad) interact most frequently with the charged O1P and O2P phosphate atoms of the DNA backbone, Dap has the specific ability to form bridges connecting neighboring phosphate groups along the sugar-phosphate backbone of the DNA strands. Also using MD simulations, Brysont and Greenall [START_REF] Brysont | Binding sites of the polyamines putrescine, cadaverine, spermidine and spermine on A-and B-DNA located by simulated annealing[END_REF] showed that electrostatic interactions concern just phosphate groups, hydrogen bonds may occur with the backbone and with DNA bases and hydrophobic interactions can result from hydrocarbon chains of diamine "binding" to non-polar parts of DNA (bases). These authors also argue that diamines may displace water from hydration sites on DNA and should therefore replace the function of DNA solvation by hydrogen bonding with amino groups. Overall, we can summarize saying that the diamines used in this study should interact similarly, i.e. showing more electrostatic DNA phosphate binding than any other type of binding.

Trihydroxymethylaminomethane (Tris)

At neutral pH, the Tris amino group is protonated, the chemical buffer cannot therefore be considered as a simple "spectator" molecule in the presence of a DNA macro-anion. Based on free solution capillary electrophoresis measurements it has been demonstrated that most of the commonly used DNA-buffers form complexes with DNA [START_REF] Stellwagen | DNA and buffers: are there any noninteracting, neutral pH buffers?[END_REF] and that Tris ions might shield DNA's phosphate groups to a similar extent to sodium ions (Na + ).

Basic amino acids:

Several studies [START_REF] Seeman | Sequence-specific recognition of double helical nucleic acids by proteins[END_REF][START_REF] Luscombe | Protein-DNA interactions: amino acid conservation and the effects of mutations on binding specificity[END_REF][START_REF] Cheng | Ab initio interaction energies of hydrogen-bonded amino acid side chain[bond]nucleic acid base interactions[END_REF][START_REF] Lancelot | Models of interaction between nucleic acids and proteins. Hydrogen bonding of arginine with nucleic acid bases, phosphate groups and carboxylic acids[END_REF][START_REF] Kono | Structure-based prediction of DNA target sites by regulatory proteins[END_REF] have shown that Arg and Lys protein residues bind predominantly to DNA bases through hydrogen bonds following a multiple donor mode of interaction; they

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both display strong specificity for the guanine base (G) in DNA. Arg and Lys residues which present more than one side-chain hydrogen-bonding atom can also, to a lesser extent, produce complex interactions with multiple bases simultaneously [START_REF] Cheng | Ab initio interaction energies of hydrogen-bonded amino acid side chain[bond]nucleic acid base interactions[END_REF][START_REF] Luscombe | Amino acid-base interactions: a threedimensional analysis of protein-DNA interactions at an atomic level[END_REF]; i.e. with two adjacent bases presenting acceptor atoms. For specific interactions, Arg and Lys strongly favor guanine (G) in hydrogen bonding, for Arg the average distribution of hydrogen bonds is for example 2.1 % C, 6.5% T, 5.1% A, 26.1% G and for non-specific interactions; 2.1 % sugar and 58.1% phosphate [START_REF] Luscombe | Amino acid-base interactions: a threedimensional analysis of protein-DNA interactions at an atomic level[END_REF]. Despite its apparent energetic advantage over Arg-G [START_REF] Cheng | Ab initio interaction energies of hydrogen-bonded amino acid side chain[bond]nucleic acid base interactions[END_REF], the Lys-G interaction is observed only one third as frequently as the Arg-G interaction. Cheng and Frankel [START_REF] Cheng | Ab initio interaction energies of hydrogen-bonded amino acid side chain[bond]nucleic acid base interactions[END_REF] have suggested that the rotational symmetry of the Lys amino group provides more ways to interact with the phosphate backbone, which therefore lowers bidentate base-specific interactions for lysine. Histidine appears to be much less reactive for both specific and non-specific interactions with DNA [START_REF] Luscombe | Amino acid-base interactions: a threedimensional analysis of protein-DNA interactions at an atomic level[END_REF] (it should be kept in mind that in its natural form, its side chain is not ionized at neutral pH). Thus, we can summarize by saying that the major interactions of basic amino acids with DNA are the following: non-specific interactions are the majority; they mainly concern the anionic phosphate sites of the DNA backbone. Specific interactions involving the three basic amino acids are of multiple natures with however a preference for the G base; Arg-G interactions are the most frequent and appear under several forms (simple, bidentate, complex). Lys-G interaction is one third as frequent as Arg-G. Other things being equal, His interactions with DNA are much less frequent than those of Arg and Lys.

In order to close this overview, we present in Fig. 2 the most frequent binding sites of the diamines and amino acid Arg to DNA. For convenience purposes and to keep the figure legible, we have limited the illustrations to the most frequent binding sites of diamines and to the only basic amino acid, arginine. Arginine is in fact by far the species forming the highest percentage of specific DNA binding, except for Van der Waals bonds with the guanine base for which Lys presented a number of bonds in slight excess of those of Arg (see [START_REF] Luscombe | Amino acid-base interactions: a threedimensional analysis of protein-DNA interactions at an atomic level[END_REF] for more details). On the other hand, Tris essentially binds to the DNA phosphate moiety and is not shown in the figure.

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Fig. 2. Two GC base-pairs with the DNA backbone are shown in blue color. The main binding sites of Arg are phosphate binding (58.1%) and bidentate binding (26.1% involving several forms of bidentate coupling to G, see for example Luscombe et al [START_REF] Luscombe | Amino acid-base interactions: a threedimensional analysis of protein-DNA interactions at an atomic level[END_REF]. for more details), the 15.8% remaining are composed of multiple bonds to other bases and to the sugar group (2.1 % C, 6.5% T, 5.1% A and 2.1 sugar). Diamines bind preferentially to the phosphate moiety of the DNA backbone with the possibility, notably for Dap, to bind two consecutive phosphate groups [START_REF] Korolev | Molecular dynamics simulation study of oriented DNA with polyamine and sodium counterions: diffusion and averaged binding of water and cations[END_REF]. Blue and red dashed lines are H-bonds; respectively intrinsic to DNA base pairing and formed between an amino acid and a base. Full red arrows indicate the possibility for diamines to bind to only one DNA strand or to connect with two different DNA helices (possibly also to connect two strands of the same closed plasmid). The red arrow which crosses the DNA double-strand indicates the special case of Dap which is able to bind two phosphate groups from opposite DNA strands across the minor groove [START_REF] Brysont | Binding sites of the polyamines putrescine, cadaverine, spermidine and spermine on A-and B-DNA located by simulated annealing[END_REF], this can also be the case for Put. With only one ammonium group, Tris essentially binds to the phosphate moiety of the DNA backbone (not shown so as not to clutter the Figure).

Thickness and composition of the layers

Nanometer-scaled layers of DNA-Ligand complexes deposited on HOPG were prepared following the protocol presented in detail elsewhere [START_REF] Boulanouar | Soft adsorption of densely packed layers of DNA-plasmid • 1,3-diaminopropane complexes onto highly oriented pyrolitic graphite designed to erode in water[END_REF]. Briefly described, the technique consists in a soft adsorption process during which a drop (50 μl) of an aqueous solution of a given plasmid DNA in equilibrium with a ligand (linker) is deposited onto freshly cleaved HOPG foil itself deposited on a 1 x 1 cm glass strip by using double-faced adhesive tape.

After 15 minutes incubation, the residual solution was gently absorbed using filter paper and the layer dried under clean air for 3 min. The layers formed were first analyzed using Atomic Force Microscopy (AFM) in order to check their surface roughness (planeity) and the homogeneity of the deposits on large areas (typically, circular deposits of 8 mm diameter were assessed by AFM over 10×10 or 5×5 µm 2 areas at least at three random positions).

Then, to measure the thickness we used the so-called "scratching technique" [START_REF] Boulanouar | Soft adsorption of densely packed layers of DNA-plasmid • 1,3-diaminopropane complexes onto highly oriented pyrolitic graphite designed to erode in water[END_REF][START_REF] Boulanouar | Dissociative electron attachment to DNA-diamine thin films: impact of the DNA close environment on the OH-and O-decay channels[END_REF].

Diamines of Eda, Dap, Put and Cad were first used in order to create thin layers of DNA complexes deposited on HOPG. Typical AFM images of such layers are presented in Fig. 3.

The plasmid (pUC21) DNA concentration was constant and equal to 20 ng.µL -1 and the mixing ratio R was fixed at 16. With diamines and R = 16 there are 16 diamines per DNA phosphate moiety in the aqueous solution drop-cast onto the graphite surface. R = 16 was chosen because we found [START_REF] Boulanouar | Soft adsorption of densely packed layers of DNA-plasmid • 1,3-diaminopropane complexes onto highly oriented pyrolitic graphite designed to erode in water[END_REF] that with increasing mixing ratios, for R > 10, in the presence of Dap, the layer thickness measured followed a plateau-like behavior at constant DNA concentration.

Close inspection of Fig. 3 As reported in a previous study [START_REF] Boulanouar | Soft adsorption of densely packed layers of DNA-plasmid • 1,3-diaminopropane complexes onto highly oriented pyrolitic graphite designed to erode in water[END_REF] where Dap was already used as a linker, when following our deposition protocol, the thickness of the layers formed can be controlled either by varying the plasmid DNA concentration and/or the mixing ratio R. Direct statistical measurements of the average thickness of the layers were performed at the fixed mixing ratio R=16 and for various plasmid concentrations (C DNA ) in the presence and absence of Tris buffer. Experimental determinations of the layer's thicknesses were repeated statistically (at least three measurements per sample) to provide a relationship that might be used to scale layers of plasmid DNA complexed with Dap and Arg in the range 5 -50 nm when deposited onto HOPG. All the results obtained are gathered in Fig. 4 and presented in Table 1. The relation h (nm) = -0.3452 + 0.1008 C DNA (R 2 = 0.9520) is the best fit to the complete data set of mean experimental layer thickness (see dashed line on Fig. 4). The general trend

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observed in the range of DNA concentrations studied is a steady increase of the thicknesses as C DNA increases. This relationship can if needed, serve to scale layers within a ± 6 nm precision using the linkers Dap and Arg with or without TE buffer being added, but always using a mixing ratio R=16. Within our experimental uncertainties, especially with a standard deviation that reached a maximum of ± 6 nm for the measured mean layer thicknesses, it can be observed that Arg and Dap essentially lead to identical thicknesses within identical experimental deposition conditions and parameters. This is particularly the case at the lower plasmid DNA concentrations (C DNA < 300 ng.µl-1), where the dispersion of experimental data appears to be minimized. When the thickness of the layers is greater than 30 nm, a larger dispersion can clearly be observed. As was previously shown [START_REF] Boulanouar | Soft adsorption of densely packed layers of DNA-plasmid • 1,3-diaminopropane complexes onto highly oriented pyrolitic graphite designed to erode in water[END_REF] in the case of layers where Dap was associated to plasmid DNA, the presence of the Tris buffer does not dramatically modify the value of the measured thickness of the layers. We show here that this is also the case for Arg. During the course of this study, we observed that the absence of the chemical buffer Tris did not alter the organization of the [plasmid DNA -linker] film's structure when using Dap or Arg. However as shown previously [START_REF] Boulanouar | Soft adsorption of densely packed layers of DNA-plasmid • 1,3-diaminopropane complexes onto highly oriented pyrolitic graphite designed to erode in water[END_REF], with Dap as a linker the presence of the Tris buffer had a strong impact on keeping a high % of supercoiled topology when the layers have to be subsequently dissolved in water (see also the next section for more details). In Fig. 5, we present two typical AFM snapshots of plasmid DNA layers were the linkers are Arg and His respectively. These pictures, when compared to those presented in Fig. 3, where the linkers are diamines, show the absence of holes in the layers and therefore the capacity of the amino acids used to provide very compact layers. It should be pointed out that, in contrast with the observations made with Arg as the linker, for the layer containing His a network of linked plasmids forming chains is observed all over the surface. The surfaces of layers made using the Lys linker are essentially identical to those made with Dap or Put.

Layers in which

When auto-organizing on the surface of HOPG during the 3-D to 2-D transition the electrostatic repulsion between plasmids must be dramatically lowered by both Arg and His linkers because holes are absent at the nanometer scale. The way in which this process arises reveals some specific features of the interaction of these amino acids with plasmid DNA, which contrast with diamines. In the latter case, it is indeed likely that the interplasmid electrostatic forces that repel the macromolecules due to excess charges are at the origin of the observed holes (Fig. 3) in the layers. In the right-hand figure where His was used, a network of complexed plasmids, which seem to be linked together to form longer chains is clearly visible over the surface of the dense layer. It should be noted that an isolated pUC21 plasmid deposited on HOPG has a length ranging from 500 to ~800 nm, depending on its topological form. For convenience, the inset (1x1 µm) shows an AFM snapshot of the same plasmids (pUC21) deposited on HOPG without any linker.

To get better insight into the structuration of the layers we analyzed a series of nanometerscaled layers containing pUC21 plasmid DNA using X-ray Photoelectron Spectroscopy (XPS).

N 1s and P 2p peaks relative to layers containing Arg, His and Lys mixed with pUC21 plasmid DNA are presented in Fig. 6. ). The left-hand spectra are relative to the XPS N1s peaks and the right-hand spectra show the XPS P2p peaks. N1s peaks are specific to the different nitrogencontaining moieties of DNA and the side-chains of the amino-acids used (see Fig. 1 and Fig. 2 for details).

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XPS analyses were also performed on layers made of pUC21 plasmids complexed with Eda and Dap (spectra not displayed herein). First, the presence of phosphorus in the XPS spectra is undeniable evidence for the presence of DNA on the substrate's surface. Second, as the sequence of the pUC21 plasmid DNA used in this study is known (provided by the supplier), we can easily determine the exact ratio (N/P) pUC21 in pure DNA plasmids. From the complete sequence of plasmid pUC21 we get (N/P) pUC21 = 3.75 per base pair. As the XPS phosphorous signal can only result from DNA, the XPS ratio (N/P) XPS measured on layers containing linkers will thus provide an estimate of the nitrogen in excess in the analyzed layer. Therefore, XPS can provide a first estimate of the number of diamines or amino acid molecules per plasmid, or better per base pair in the analyzed layers. To explain the method let us call  the number of nitrogen atoms found in excess per phosphate atom ( = (P/N) XPS -3.75) and  the number of nitrogen atoms per ligand molecule (see Fig. 1). The number of ligand molecules per base pair (i.e. per two phosphate moieties) will thus be 2/. The number  of ligand molecules which are in excess expressed in (bp -1 ) is thus ( = 2/where  corresponds to the maximum number of ligand molecules per base pair at a hypothetical complete saturation. We set thus  = 2 for diamines and  = 3 for amino acids (for more details see the section dealing with binding sites). The value  = 3 for amino acids is in fact an overestimate because it implies that the DNA plasmid would be completely composed of GC base pairs; those with which Arg contacts are the most frequent. It should also be kept in mind that the composition estimated from an XPS peak area analysis is accurate within ~10%-20% [START_REF] Tougaard | Validity of automated x-ray photoelectron spectroscopy algorithm to determine the amount of substance and the depth distribution of atoms[END_REF] depending on the particular depth distribution of the atoms. Overall, the  values we will determine are thus estimates. We present, in Table 2, the results in terms of  values obtained by means of XPS analyses for layers containing Eda, Dap, Arg, His and Lys linkers and for which R = 16 and C DNA = 200 ng.µL -1 . )

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0 0 ~7 ~4 ~1
It appears clearly from Table 2 that linkers (ligands) which are in excess as compared to a full saturation of the potential binding sites, are the basic amino acids Arg, His and to a much lesser extent Lys. Eda is found to be at a balance of ~1 per bp in the layers. Dap provides a quasi-complete saturation in terms of phosphate moieties of the DNA backbone (~2 per base pair). In contrast, Arg and His appear to be in large excess in the layers. This observation therefore suggests that these two basic amino acids may have specific properties, which make it possible to incorporate large additional proportions of such ionized (positivelycharged) molecules into the plasmid DNA layers.

Recovery of complex films in water and conservation of supercoiling

The layers described above have the original property of being soluble in water, except, the part (first monolayer) in close contact with the HOPG surface which remains strongly linked to the HOPG, as previously reported [START_REF] Boulanouar | Soft adsorption of densely packed layers of DNA-plasmid • 1,3-diaminopropane complexes onto highly oriented pyrolitic graphite designed to erode in water[END_REF] for Dap. In order to check the suitability of the layers for further experiments where they may be re-dissolved in water, we re-suspended in water layers for which the linkers were Dap, Put, Arg, His and Lys and we statistically determined the mean percentage of supercoiled topology at the end of the complete process (i.e. complexation in aqueous solution, deposition on the HOPG surface, further dissolution in water). The % of supercoiled topology is determined by means of standard agarose gel electrophoresis analyses. Eda is not used in that part of the experiments due to the poor quality of the layers it provides (high porosity); this observation by the way seems to be confirmed by its low -value (Table 2). On the other hand, Put was added to the set of ligands incorporated in the layers because it provides layers with surface properties comparable to those containing Dap (Fig. 3). In order to illustrate the role of the Tris buffer in maintaining the supercoiled topology over all the different steps; experiments were performed both in the presence and in the absence of Tris. The complete set of results is presented in Fig. 7. It clearly appears that the amino acids Arg and His, are best able to maintain the supercoiled topology of pUC21 plasmid DNA after the various steps described above have been followed; this is particularly clear when the Tris buffer is absent. Between the two amino acids, Arg is the better with only 2% loss of supercoiling in the presence of Tris buffer (we measured an average of 97% supercoiled DNA without linker in aqueous solution and 95 % for the re-suspended mixture with Arg in the presence of Tris). When no buffer is added, 9% of the supercoiled topology is lost, which is rather good if additional experimental steps have to be added to the process and where the supercoiled fraction might finally be assessed. With His, in the presence of Tris, the supercoiled fraction drops down to 89% (8% variation) and in its absence 15% of the supercoiled fraction is lost. As may be anticipated, Put and Dap which essentially bind DNA with equivalent features (predominantly via non-specific interactions), provide comparable data. Within identical experimental conditions, Lys, even though it binds to DNA through both specific and nonspecific interactions, leads to rather high losses of supercoiling (comparably to the diamines Dap and Put).

Discussion

In this study, we compared the ability of the basic amino acids Arg, His and Lys with those of diamines Eda, Dap, Put and Cad to form dense, nanometer-scaled recoverable layers when complexed with plasmid DNA and deposited onto freshly cleaved surfaces of HOPG. All the species mentioned form layers with plasmid DNA which can afterwards be resuspended in water by simple washing. A bibliographic survey has shown that the basic amino acids Arg and Lys have a particular propensity to develop specific interactions (i.e. with the DNA bases) in much larger proportions than all other linkers studied, including His.

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Diamines Eda and Cad, respectively the shortest and the longest diamines used herein, produced layers with large holes when observed at the nanometer scale. This specific property is likely to be in relation with the sizes of the linkers (namely Eda and Cad); the length of latter does not match the distance that separates two consecutive phosphate moieties along the DNA backbone, or even the distance that separates two phosphates on opposite strands of a same DNA macromolecule. This results in dangling excess positive charges, likely to generate electrostatic repulsion between DNA plasmids, which will not thus, for the most part, be neutralized. Dap and Put, whose average nitrogen-nitrogen distances are close to the distance that separates two consecutive, or opposite phosphate moieties along the DNA backbone form much denser complex layers.

The case of the basic amino acids Arg, His and Lys is different in principle, due the zwitterionic character of their main chain. In addition, these three amino acids each have a particular terminal positively-charged side chain, composed of a guanidinium, an imidazole and an amino moiety when dealing respectively with Arg, His and Lys. Deposits in which Arg and His are the linkers, when analyzed by AFM, showed very high surface compacity; holes

were not observed at the nanometer scale. The fact that Arg and Lys are the two linkers that form a high proportion of specific interactions with DNA [START_REF] Seeman | Sequence-specific recognition of double helical nucleic acids by proteins[END_REF] cannot be invoked as the sole reason to explain the protective effect observed here, and which is anyway significantly higher when using Arg than Lys. On the other hand, we have shown that Arg and His are the linkers that remain in the greatest proportions in the layers (XPS analyses of the composition of the layers). It indeed seems likely that the greater the amount of the linkers remaining in excess in a layer (so-called -values), the higher the protective effect against loss of supercoiling. In this regard, recent molecular dynamics simulations that concur with this last hypothesis show significant like-charge pairing of guanidinium side chains in aqueous polyarginine [START_REF] Vondraśěk | The molecular origin of likecharge Arginine-Arginine pairing in water[END_REF][START_REF] Vazdar | Like-charge guanidinium pairing from molecular dynamics and ab initio calculations[END_REF], while such an effect is absent in aqueous poly-lysine. Interestingly too, other recent studies based on numerical simulations have reported mesoscopic structures arising from aggregates or clusters of 1,3-dialkylimidazolium-based compounds at room temperature in ionic liquids [START_REF] Turton | Dynamics of imidazolium ionic liquids from a combined dielectric relaxation and optical Kerr effect study: evidence for mesoscopic aggregation[END_REF] but also in the aqueous phase for the special case of histidine-histidine [START_REF] Heyda | Attractive interactions between side chains of Histidine-Histidine and Histidine-Arginine-based cationic dipeptides in water[END_REF]. These last observations are in turn in favor of an aggregation of histidine molecules in solution giving rise to cation-stacking clusters. Thus, based on these observations, we may argue that within an excess number of ~7 Arg per base pair and ~4 His per base pair against only ~1 for Lys and 0 for Dap and Eda, the high protective effect

A C C E P T E D M A N U S C R I P T measured after dissolution of the layers in water should indeed be related to the excess of linker in the layers. Excess numbers of amino acids expressed per base pair greater than 1 are in fact unexpectedly high. Actually, in the theory of DNA condensation in solution (the counterion condensation concept) [START_REF] Jr | Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity[END_REF]; , namely the number of thermodynamically released Na + ions per phosphate upon ligand binding, is  ≈ 0.88 (for linear helical DNA).

Thus, those large amounts of positively-charged Arg and His molecular clusters that remain in excess in the layers might be seen as a kind of charged aggregate which binds DNA and which may even allow any holes in the layers to be filled. We may thus anticipate that when complex structures of [plasmid DNA -linkers] are formed in solution, first the oppositely charged molecules neutralize each other, reducing the net charge and consequently reducing their solubility. In parallel, aggregates of Arg or His are likely to bind DNA to form complex structures which finally precipitate on the HOPG surface. This may explain the very high compaction of the two layers observed in Fig. 5, where Arg and His are the linkers and for which holes in the layers are completely absent at the nanometer scale.

Surprisingly, at a constant mixing ratio R=16, the dependence of the average thickness of plasmid deposits containing Arg and Dap follows within a maximum ± 6 nm deviation the same trend when plotted versus DNA concentration (in the 0 -500 ng.µL -1 concentration range). A simple fit to the experimental data provides a scaling relation that may be useful to all those who wish to use well-gauged nanometer-scaled plasmid DNA layers containing Dap or Arg deposited on HOPG with the property of being able to be re-dissolved in water. In addition, the relationship we provide seems to be invariant depending on whether the buffer Tris is present or absent. This is actually a very interesting point when dealing with electron stimulated desorption (ESD) of DNA samples [START_REF] Boudaiffa | Resonant formation of DNA strand breaks by low energy (3-20 eV) electrons[END_REF][START_REF] Boulanouar | Dissociative electron attachment to DNA-diamine thin films: impact of the DNA close environment on the OH-and O-decay channels[END_REF], a refined technique which helps understanding and describing the fundamental aspects of radiation-induced damage of DNA.

When using ESD, any additional chemical species present in the analyzed sample (layer) might create additional desorption signals and therefore might interfere and engender noisy signals. As ESD studies do not generally require further gel electrophoresis analysis, the protocol exposed herein may be extremely useful in such research areas in relation with fundamental radiobiology. Such layers, containing Tris or not, will also most probably find applications in areas where plasmid DNA deposits are used for the controlled release of therapeutic macromolecules [START_REF] Yamauchi | Layer-by-layer assembly of poly(ethyleneimine) and plasmid DNA onto transparent Indium-Tin oxide electrodes for temporally and spatially specific gene transfer[END_REF][START_REF] Hujaya | Multilayered thin films from poly(amido amine)s and DNA[END_REF]. Lastly, when present in the solution, the Tris buffer undoubtedly ensures conservation of a maximum percentage of supercoiled topology of the

A C C E P T E D M A N U S C R I P T

DNA plasmids after further recovering in water. Adding this buffer is thus a necessary condition for keeping high plasmid supercoiled topology when Dap, Put or Lys are used as linkers.

Conclusion

All the ligands used allowed ultra-thin and water-soluble plasmid DNA layers to be made on and Dap) shows that the higher the excess of ligand molecules in the layer, the greater the protection of the supercoiled topology of the plasmid DNA throughout the various steps of the experimental procedure. Arginine provided the best protection against single-strand breakage (i.e. loss of the supercoiled topology), even in the absence of the Tris buffer, only 9% of supercoiled topology is lost. This result is consistent with the fact that in eukaryotic cells, arginine is the most frequent binding protein tail in histones (namely in histones H3 and H4 among the 5 main histone proteins).

Well-gauged layers (with thicknesses ranging from 10 to 50 nm) of pUC21 plasmid DNA mixed with Dap have very recently been used in experiments aiming to determine the mean effective attenuation length (EAL) of low energy electrons (energies < 20 eV) in DNA by exposure to electrons in air [START_REF] Fromm | Low energy electrons and ultra-soft X-rays irradiation of plasmid DNA. Technical innovations[END_REF]. Besides key experimental determinations of fundamental parameters such as EALs or damage cross-sections [START_REF] Boulanouar | Absolute cross section for loss of supercoiled topology induced by 10 eV electrons in highly uniform /DNA/1,3diaminopropane films deposited on highly ordered pyrolitic graphite[END_REF], the use of ultra-thin plasmid DNA layers such than those presented and characterized herein might also be useful in other recent research and development areas such as for example DNA thin film sensors for alpha radiation [START_REF] Kulkarni | A novel nanometric DNA thin film as a sensor for alpha radiation[END_REF] or Bio-Organic Optoelectronic Devices [START_REF] Singh | Bio-organic optoelectronic devices using DNA[END_REF].

Fig. 1. Linear diamines: ethylene diamine (Eda), 1,4-diaminobutane (putrescine or Put), 1,5-diaminopentane (cadaverine or Cad) as well as basic amino acids histidine (His), arginine (Arg) and lysine (Lys) Protonated amino groups which contribute to a bulk positive charge of the molecules at neutral pH are in bold blue. Due to triple resonance stabilization, the guanidinium group of the arginine (Arg) amino acid presents 3 potentially positively-charged nitrogen atoms [START_REF] Schug | Noncovalent binding between Guanidinium and anionic Groups: focus on biological-and synthetic-based Arginine/Guanidinium interactions with phosph[on]ate and sulf[on]ate residues[END_REF]. [START_REF] Luscombe | Amino acid-base interactions: a threedimensional analysis of protein-DNA interactions at an atomic level[END_REF]. for more details), the 15.8% remaining are composed of multiple bonds to other bases and to the sugar group (2.1 % C, 6.5% T, 5.1% A and 2.1 sugar). Diamines bind preferentially to the phosphate moiety of the DNA backbone with the possibility, notably for Dap, to bind two consecutive phosphate groups [START_REF] Korolev | Molecular dynamics simulation study of oriented DNA with polyamine and sodium counterions: diffusion and averaged binding of water and cations[END_REF]. Blue and red dashed lines are H-bonds; respectively intrinsic to DNA base pairing and formed between an amino acid and a base. Full red arrows indicate the possibility for diamines to bind to only one DNA strand or to connect with two different DNA helices (possibly also to connect two strands of the same closed plasmid). The red arrow which crosses the DNA double-strand indicates the special case of Dap which is able to bind two phosphate groups from opposite DNA strands across the minor groove [START_REF] Brysont | Binding sites of the polyamines putrescine, cadaverine, spermidine and spermine on A-and B-DNA located by simulated annealing[END_REF], this can also be the case for Put. With only one ammonium group, Tris essentially binds to the phosphate moiety of the DNA backbone (not shown so as not to clutter the In the right-hand figure where His was used, a network of complexed plasmids, which seem to be linked together to form longer chains is clearly visible over the surface of the dense layer. It should be noted that an isolated pUC21 plasmid deposited on HOPG has a length ranging from 500 to ~800 nm, depending on its topological form. For convenience, the inset (1x1 µm) shows an AFM snapshot of the same plasmids (pUC21) deposited on HOPG without any linker. ). The left-hand spectra are relative to the XPS N1s peaks and the right-hand spectra show the XPS P2p peaks. N1s peaks are specific to the different nitrogencontaining moieties of DNA and the side-chains of the amino-acids used (see Fig. 1 and Fig. 2

Basic amino acids

for details).

Binding Energy (eV)

4 -].

 4 diaminobutane dihydrochloride (≥98%), 1,5-diaminopentane dihydrochloride (≥98%) and protonated basic amino acids of arginine hydrochloride (≥98%), histidine hydrochloride (≥98%), and lysine hydrochloride (≥98%) were all purchased from Sigma-Aldrich (France) and kept at 4 • C. Plasmid DNA (pUC21, 3266bp length and with a molecular weight of 2.13 x 10 6 Da) at an initial concentration of 1 mg/mL in water for injection (WFI) was purchased from Plasmid Factory GmbH & Co. KG (Germany). The pUC21 mother solution was free of Tris buffer. Without further purification, stock solutions of this DNA (>95% supercoiled) were prepared by dilution with ultrapure water having a resistivity of 18.2 MΩ cm. The stock solutions were stored at -20°C. DNA concentrations were determined by measuring the absorbance at 260 nm using the molar extinction coefficient ε 260 = 5.3 × 10 7 cm -1 M -1 .Solutions of plasmid DNA were mixed with an equal volume of a solution containing protonated ligand molecules to attain the desired ratios R = 16 at a final DNA concentration (C DNA ) in the range 100 -500 ng.µL -1 . R, the mixing ratio, is defined as the ratio of the molar concentration of the cations to that of anionic phosphate moieties of plasmid DNA in the solution, namely R = [cation]/[PO When the Tris buffer had to be present in the solution, the pUC21 plasmid DNA mother solution was first diluted in Tris/EDTA so that the final concentration of Tris is 10 mM Tris-HCl and 1 mM EDTA, i.e. pH = 7.6. Solutions were freshly prepared before each experiment.

  clearly shows that the nanoscale surface roughness of the samples can be separated into two trends with Eda and Cad (n= 2 and n=5) in one group and Dap and Put (n=3 and n=4) in the other. Holes (porosity) are indeed much greater in the deposits A C C E P T E D M A N U S C R I P T containing Eda and Cad than in those containing Dap and Put. Dap appears to be the linker which provides the most compact layers. Besides, if we compare the average thickness Z (nm) of the layers (right-handed part of Fig. 3) all layers are similar, in the range 4-6 nm.

Fig. 3 .

 3 Fig. 3. AFM images (5x5 µm) of layers composed of plasmid DNA complexed to Eda, Dap, Put and Cad deposited on HOPG (R= 16, C DNA =20 ng/μl). The grey horizontal segments indicate the lines that where scanned to obtain the right-handed panel where the surface roughness is measured versus distance. Estimates of the layer thickness (nm) remain close together, their average value being 4.75±0.96 nm.

Fig. 4 . 1 )

 41 Fig. 4. Average thickness measurements (AFM) performed over at least three fields (5x5 µm) for various types of [pUC21-linker] layers (error bars represent the standard deviations). The data at C DNA = 20 ng.µL -1 are rough estimates (see text). Layers containing Dap (except the one at C DNA = 20 ng.µL -1 ) and Arg were prepared in the presence or absence of Tris-EDTA buffer (TE). The dashed line constitutes the best linear fit over all experimental points (R 2 = 0.95). The inset at the bottom right is an example of the accurate AFM thickness (h) measurement ("scratching technique") used and concerns the highest right-handed pale blue diamond. Except for the data set at C DNA = 20 ng.µL -1 all thicknesses were measured using this accurate technique.

  Lys and His have been used as linkers were also imaged by AFM. Accurate measurements of the thicknesses of the layers were performed only for some DNA concentrations; namely with Lys in the presence of Tris at C DNA = 200 and C DNA = 300 ng.µL -1 (R = 16 ); the thicknesses found were h = 8.3 ±4 nm and h = 21 ±3 nm respectively. These two thicknesses appear to be somewhat lower than those obtained when Dap or Arg were used and that is why we did not add the data concerning Lys to Fig. 4. As for His, in the presence A C C E P T E D M A N U S C R I P T of Tris, a series of three thickness measurements performed with C DNA = 200 ng.µL -1 and R = 16 provided an average value h = 17 ± 2 nm in good agreement with what was measured in the presence of Dap or Arg, at the same plasmid DNA concentration.

Fig. 5 .

 5 Fig. 5. AFM images (5x5 µm) of layers composed of plasmid DNA complexed to (left) Arg and (right) His. On the left-hand figure, it appears that Arg is the linker which enables the highest density of the layers to be obtained.

Fig. 6 .

 6 Fig. 6. XPS spectra (counts in arbitrary units, a.u.) of Arg, His and Lis complexed to pUC21 plasmid DNA and deposited on freshly cleaved HOPG(R=16 and C DNA = 200ng.µL -1

Fig. 7 .

 7 Fig. 7. Histograms of the average % of supercoiled topology as measured on DNA-linker mixtures obtained following dissolution of the layers in ultra-pure water. Error bars are the standard deviations determined for three measurements made on aliquots. TE stands for Tris-EDTA buffer.

  HOPG. Due to the fact that in diamines of Dap and Put, ammonium-ammonium separation distances are close to the distance that separates two consecutive phosphate moieties in DNA, dense layers characterized by weak porosity can be produced. In contrast, the layers obtained by using Eda and Cad exhibit large holes at the nanometer scale. Although they have different chemical structures, the basic amino acids Arg, His and Lys provide selfassembly of plasmid DNA layers with similar structural properties. Statistical measurements of the mean heights of various layers containing ligands of Dap and Arg show that the thicknesses of the layers have the same linear dependence when plotted versus DNA concentration at fixed mixing ratio R = 16, within a ± 6 nm maximum deviation. In addition, this relation holds whether or not Tris is present in the drop-cast solution. The relation we provide may come in handy in experiments in which surface roughness and thickness have to be accurately controlled (i.e., at the nanometer scale).Considering that the limit of ligand binding to DNA is reached for a complete saturation of the binding sites expressed per base pair, XPS data provides an estimate of the excess ligand molecules per base pair in a given layer. Only two ligands appear to be in large excess in the layers formed, namely Arg and His. The excess of ligands in the layers decreases in the following way Arg > His >> Lys > Dap > Eda. When determining the residual fraction (in %) of supercoiled plasmid DNA present in the solution resulting from recovery of the layers in water, the following sequence is obtained Arg > His >> Lys > Dap > Put, whether Tris was present or absent from the drop-cast solution. The one-to-one relationship (for Arg, His, Lys

Fig. 2 .

 2 Fig. 2.Two GC base-pairs with the DNA backbone are shown in blue color. The main binding sites of Arg are phosphate binding (58.1%) and bidentate binding (26.1% involving several forms of bidentate coupling to G, see for example Luscombe et al[START_REF] Luscombe | Amino acid-base interactions: a threedimensional analysis of protein-DNA interactions at an atomic level[END_REF]. for more details), the 15.8% remaining are composed of multiple bonds to other bases and to the sugar group (2.1 % C, 6.5% T, 5.1% A and 2.1 sugar). Diamines bind preferentially to the phosphate moiety of the DNA backbone with the possibility, notably for Dap, to bind two consecutive phosphate groups[START_REF] Korolev | Molecular dynamics simulation study of oriented DNA with polyamine and sodium counterions: diffusion and averaged binding of water and cations[END_REF]. Blue and red dashed lines are H-bonds; respectively intrinsic to DNA base pairing and formed between an amino acid and a base. Full red arrows indicate the possibility for diamines to bind to only one DNA strand or to connect with two different DNA helices (possibly also to connect two strands of the same closed plasmid). The red arrow which crosses the DNA double-strand indicates the special case of Dap which is able to bind two phosphate groups from opposite DNA strands across the minor groove[START_REF] Brysont | Binding sites of the polyamines putrescine, cadaverine, spermidine and spermine on A-and B-DNA located by simulated annealing[END_REF], this can also be the case for Put. With only one ammonium group, Tris essentially binds to the phosphate moiety of the DNA backbone (not shown so as not to clutter the Figure).

  Fig. 2.Two GC base-pairs with the DNA backbone are shown in blue color. The main binding sites of Arg are phosphate binding (58.1%) and bidentate binding (26.1% involving several forms of bidentate coupling to G, see for example Luscombe et al[START_REF] Luscombe | Amino acid-base interactions: a threedimensional analysis of protein-DNA interactions at an atomic level[END_REF]. for more details), the 15.8% remaining are composed of multiple bonds to other bases and to the sugar group (2.1 % C, 6.5% T, 5.1% A and 2.1 sugar). Diamines bind preferentially to the phosphate moiety of the DNA backbone with the possibility, notably for Dap, to bind two consecutive phosphate groups[START_REF] Korolev | Molecular dynamics simulation study of oriented DNA with polyamine and sodium counterions: diffusion and averaged binding of water and cations[END_REF]. Blue and red dashed lines are H-bonds; respectively intrinsic to DNA base pairing and formed between an amino acid and a base. Full red arrows indicate the possibility for diamines to bind to only one DNA strand or to connect with two different DNA helices (possibly also to connect two strands of the same closed plasmid). The red arrow which crosses the DNA double-strand indicates the special case of Dap which is able to bind two phosphate groups from opposite DNA strands across the minor groove[START_REF] Brysont | Binding sites of the polyamines putrescine, cadaverine, spermidine and spermine on A-and B-DNA located by simulated annealing[END_REF], this can also be the case for Put. With only one ammonium group, Tris essentially binds to the phosphate moiety of the DNA backbone (not shown so as not to clutter the Figure).

Fig. 3 .Fig. 4 . 1 )Fig. 5 .

 3415 Fig. 3. AFM images (5x5 µm) of layers composed of plasmid DNA complexed to Eda, Dap, Put and Cad deposited on HOPG (R= 16, C DNA =20 ng/μl). The grey horizontal segments indicate the lines that where scanned to obtain the right-handed panel where the surface roughness is measured versus distance. Estimates of the layer thickness (nm) remain close together, their average value being 4.75±0.96 nm.

Fig. 6 .

 6 Fig. 6. XPS spectra (counts in arbitrary units, a.u.) of Arg, His and Lis complexed to pUC21 plasmid DNA and deposited on freshly cleaved HOPG(R=16 and C DNA = 200ng.µL -1

Figure 7 :

 7 Figure 7: Histograms of the average % of supercoiled topology as measured on DNA-linker mixtures obtained following dissolution of the layers in ultra-pure water. Error bars are the standard deviations determined for three measurements made on aliquots. TE stands for Tris-EDTA buffer.

Table 1 .

 1 Experimentally determined mean thicknesses and standard deviations for pUC21 plasmid DNA layers complexed with Dap and Arg linkers on HOPG; R = 16.

	pUC21 concentration (ng.µL	) -1	Dap	Dap (TE)	Arg (TE)	Arg
	100		10(±2)	3 (±1)	3 (±1)	7 (±3)
	200		18(±2)	19 (±1)	17 (±3)	20 (±6)
	300		27(±3)	30 (±2)	30 (±2)	25 (±5)
	400		ND	33 (±3)	ND	45 (±6)
	500		ND	58(±5)	ND	50 (±4)

*(TE) means that Tris-EDTA buffer was added to the solutions prior to the deposition process.

Table 2 .

 2 Experimentally determined (XPS) compositions of plasmid DNA layers complexed with Eda, Dap, Arg, His and Lys linkers on HOPG; R = 16. TE buffer was absent in those layers during XPS experiments.

	Ligand		Eda	Dap	Arg	His	Lys
	(N / P) pUC21		3.75	3.75	3.75	3.75	3.75
	(N / P) XPS		4.63	5.50	24.51	14.04	7.87
	 = N atoms in excess	0.88	1.75	20.83	10.29	4.12
	(per phosphate)
	 = N atoms per ligand	2	2	4	3	2
	Ligand molecules (bp	-1 )	0.88	1.75	10.42	6.86	4.12
	 = Ligand molecules in excess (bp -1

Acknowledgments: This work was partially supported by a grant from the Iraqi embassy (Ph. D. of Dr. T. T. Khalil). We are particularly indebted to Dr. Peter Winterton (Toulouse University, France) for correcting and improving the language of the present article.

A C C E P T E D

M A N U S C R I P T

Highlights:



Characterization of nanometer scaled layers composed of pUC21 plasmid DNA  Relation between nature of the ligand and structure of the layers  Capacities of the ligands to protect plasmids from strand break depending on their nature