DNA Base Modifications Mediated by Femtosecond Laser-Induced

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

DNA Base Modifications Mediated by Femtosecond LaserInduced Cold Low Density Plasma in Aqueous Solutions. Hakim Belmouaddine, Guru S Madugundu, James Richard Wagner, Arnaud Couairon, Daniel Houde, and Leon Sanche J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.9b00652 • Publication Date (Web): 30 Apr 2019 Downloaded from http://pubs.acs.org on May 1, 2019

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The Journal of Physical Chemistry Letters

DNA Base Modifications Mediated by Femtosecond Laser-Induced Cold Low Density Plasma in Aqueous Solutions Hakim Belmouaddine,∗,† Guru S. Madugundu,† J. Richard Wagner,† Arnaud Couairon,‡ Daniel Houde,† and Leon Sanche∗,† †Department of Nuclear Medicine and Radiobiology, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada. ‡CPHT, CNRS, Ecole polytechnique, IP Paris, F-91128 Palaiseau, France. E-mail: [email protected]; [email protected] Phone: +1819-346-1110 ext. 74345. Fax: +1819-564-5442

Graphical TOC Entry

Abstract Applications based on near-infrared femtosecond laserinduced plasma in biological materials involve numerous ionization events that inevitably mediate physicochemical effects. Here, the physical chemistry underlying the action of such plasma is characterized in a system of biological interest. We have implemented wavefront shaping techniques to control the generation of laser-induced low electron density plasma channels in DNA aqueous solutions, which minimize the unwanted thermo-mechanical effects associated with plasma of higher density. The number of DNA base modifications per unit of absolute energy deposited by such cold plasma are compared to those induced by either ultraviolet or standard ionizing radiation (γ-rays). Analyses of various photo-induced, oxidative and reductive DNA base products show that the effects of laser-induced cold plasma are mainly mediated by reactive radical species produced upon the ionization of water, rather than by the direct interaction of the strong laser field with DNA. In the plasma environment, reactions among densely produced primary radicals result in a dramatic decrease in the yields of DNA damages relative to sparse ionizing radiation. This intense radical production also drives the local depletion of oxygen.

ACS Paragon Plus Environment 1

H3O+

e-aq O2

.OH

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In biomedical fields, near-infrared (NIR) femtosecond (fs) lasers are, depending on irradiation parameters, used for different applications, including visualization, modification and/or manipulation of systems of biological interest. NIR fs lasers had paved the way for many advances ranging from imaging, e.g. multi-photon microscopy 1–3 , to ablation in living organisms 4,5 , e.g. nano-surgery 6 . This breadth of applications reflects the numerous different irradiation regimes in which fs lasers can interact. Among these various regimes, there exists one which maximizes the photon-induced ionization of water, when the fs laser beam propagates through aqueous biological environments, but minimizes the thermomechanical effects generally associated with the interaction with intense fs laser pulses. This specific regime of irradiation corresponds to the generation of low electron density plasma. Unlike plasma with higher electron densities, above the threshold for optical breakdown of water ρc = 1.8×1020 e− .cm−3 7 , these low-density plasma (LDP) remain relatively cold, i.e. ∆T ∼ 0.5 K 8 (also see supplementary information (SI) for details). Such conditions allow physico-chemical effects in systems of biological interest, that derive from the generation of plasma of electron densities close to threshold for optical breakdown, to be studied. These LDP have recently emerged as a new tool for studying the action of ionizing radiations in biological media 9,10 . In the latter, spatial resolution achievable with fs laser pulses allows highly localized energy deposition down to nanometer scale volumes 6 and the investigation of the consequences of ionizing irradiation at sub-cellular levels 11–14 . However, the physico-chemical effects, which underlie the biological action mediated by these LDP, remain poorly characterized. To extend the plasma generation into macroscopic volumes and study its effects in aqueous solutions, previous studies showed that the non-linear optical propagation of powerful fs laser pulses 15–17 can induce the self-regulated generation of spatially homogeneous lowdensity plasma spots in water through laser filamentation 18,19 . Here, the low-density plasma, shaped as channels, generate high rates of ionization (1018 e− .cm−3 ) in water over distances of several cm along the laser propagation axis, within radial dimensions of 5.5 µm 19,20 . We exploited the multi-filamentation of 800 nm fs laser pulses 21–23 to explore the radiation-assisted chemistry associated with the LDP generation in aqueous solutions. A major advance towards this goal has been the deployment of a spatial light modulator (SLM) to control the filamentation process 24–26 . The latter allows us to obtain a programmable matrix of mono-filaments (Figure 1.A) that provides a more homogeneous and controlled energy deposition than is possible with the randomly entangled LDP channels, or filaments, that are typically produced during the non-linear propagation of the laser beam (Movie S1) 27–30 . Using the present irradiation method, we irradiated aqueous solutions containing isolated calf-thymus

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DNA. Enzymatic digestion of the polymers releases the bases as nucleosides 31 (see SI appendix for details). We employ liquid chromatography coupled to atmospheric-pressure-ionization tandem mass spectroscopy (LC-MS/MS) analysis to identify and quantify radical-mediated DNA base modifications. Based on precise analytical chemistry, our results detail the complex laser-induced processes from the absorption of energy to the formation of DNA degradation products. These results are compared to those from 266 nm fs laser pulses and a conventional source of ionizing radiation (60 Co, γ-rays). We implement on the SLM a programmable matrix of 8x8 juxtaposed micro-lenses. Each micro-lens samples and collimates a portion of the incident laser beam so that it carries just enough power to ensure that : i) the beam collapses after 1 cm of non-linear propagation, i.e. self-focusing 16,32 , and ii) the generation of LDP is restricted to a single channel per micro-lens, i.e. a mono-filament (see SI appendix for details) 33 . Thus, to generate the LDP though the entire volume of interest, i.e. 3 mL of DNA aqueous solution in a 10x10x56 mm quartz cell, we placed, immediately in front of the sample to be irradiated, a second quartz cell filled with pure water (Figure S1). Propagation of the fs laser pulses in the first of these two cells, initiates self-focusing and ensures laser filamentation throughout the sample volume (Figure 1). The refresh rate of the SLM (60 Hz) allowed us to translate the matrix position by 100 µm every 17 ms and scan approximately 1 cm3 of aqueous solution with the laser-induced LDP channels (Movie S2). The laser repetition rate was set at 125 Hz to ensure that at least one and no more than two laser pulses irradiate the same volume of fresh solution (Figure S5). We support our experimental measurement with numerical simulations of laser filamentation in water of the fs laser pulse collimated by one of the micro-lens displayed on the SLM (see SI appendix for details). Numerical simulations supply information about the spatial distribution of the intensity of light (Figure 1.C) and the electronic density of the plasma channel (Figure 1.B) in water, along the laser propagation axis. Results obtained from the simulation show the formation of a LDP channel after ∼ 1 cm of propagation and along a second centimeter of propagation in water. Similarly, the same parameters of laser irradiation allow us to experimentally observe the white-light generation, which is spatially concomitant with laser filamentation 15,34 , but only after ∼ 1 cm of propagation in water (Figure 1.A). Rigorous comparisons between the biological effects mediated by the laser-induced LDP and the gamma irradiation requires a relevant observable value to quantitatively standardize the irradiation. In radiation science, this value usually corresponds to the dose, i.e. the absolute quantity of energy deposited per unit of mass in Gray (J.kg−1 ) 35,36 . However, this concept of dose is somewhat ill suited to fs laser irradiation. We find that the absolute quantity of energy deposited in the


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The Journal of Physical Chemistry Letters A. B.

D.

C.

Energy deposition rate (nJ/mm)

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100 80 60 40 20 0

0.50

1.00

z (cm)

1.50

2.00

Figure 1: A. Cross-sectional image at the output of the irradiated solutions of the matrix of the laser beams collimated by the micro-lenses displayed on the spatial light modulator, with the typical white-light generation, after propagation through two quartz cell of length 1 cm filled with pure water. The disparity between the images of the spots nearby the center of the matrix and the outermost ones is due to typical optical aberrations introduced by the 4-f setup. Removing the second quartz cell extinguishes the white-light. Numerical simulation of the filamentation of a 800 nm, 75 fs (Fourier limited pulse duration of 35 fs with a negative chirp), 2.35 µJ laser pulse collimated by a micro-lens (N.A. = 3.3×10−3 ) in a 2 cm long volume of water placed at the focus : B. spatial distribution of the plasma electron density; C. spatial distribution of light intensity; the green curves in figures B and C illustrate the laser beam full width at half maximum; D. Spatial distribution of the absolute energy deposition. aqueous solution of interest per unit of irradiation time, establishes the best common ground between laser and gamma irradiation. The calibrated gamma irradiator, which delivers 0.8 Gy.min−1 , deposits 2.4 mJ.min−1 in 3 mL of aqueous solutions. We evaluated the quantity of energy deposited by the laser irradiation in the same solution by measuring the difference in power of light transmitted through the aqueous solutions in which the collimated laser beams propagate, with and without LDP generation (see SI appendix for details). Numerical simulations of the spatial distribution of energy deposition in water (Figure 1.D), along the laser propagation axis, returns 76 ± 7 mJ.min−1 of energy deposited by the matrix of filaments in the second centimeter of water, in very good agreement with 59 ± 6 mJ.min−1 measured experimentally. The plasma-mediated effects on the components of an irradiated system of biological interest is supposedly to be mainly indirect, principally mediated by the low energy (0-15 eV) electrons 8,37,38 and the reactive species produced upon the laser-induced lysis of water 28,29,39,40 : nhγ

• + 2H2 O(aq) → e− aq + OH + H3 O

(1)

• where e− aq is the hydrated electron, OH the hydroxyl + radical, and H3 O the hydronium ions. As a preliminary study, we characterized the effects mediated by the programmable matrix of laser filament on compounds in water. To do this, we studied the radiation-assisted oxidation of ferrous cations Fe2+ into ferric cations Fe3+ in super-Fricke aqueous solutions irradiated by the controllable LDP channels (see SI appendix for details). We found that each of our controlled mono-filament of light mediates very similar effects, relative to each other, in the irradiated solutions (Figure S4). This preliminary study establishes that the laser-induced LDP channels comprising the controllable matrix can be considered as independent, yet identical, micro-beams of ionizing radiation. Results obtained while irradiating samples with the whole matrix can be directly extrapolated to a unique plasma channel generated by one laser pulse. Since DNA molecules strongly absorb ultra-violet light around 260 nm, we were concerned about the possibility of multi-photon absorption events. Using 800 nm high intensity light, the simultaneous interaction of three photons, equivalent to one 266 nm photon, could be problematic within the framework of our study 8,12,41 .



Table 1: Yield of photo-products in modifications per 106 DNA bases per Joule of deposited energy by the 800 nm fs laser pulses third harmonic (266 nm) and laser-induced LDP channels in aqueous solutions of isolated DNA.

fs laser-induced LDP 0.10±0.01 0.055±0.007 0.005±0.001 B/1

50 40 30 20 10 0

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Lesions / 104 DNA bases

A/1 40 30 20 10 0

Energy deposition (J)

0

2

17.5 15.0 12.5 10.0 7.5 5.0 2.5 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7


4

6

8

10 12 14

8

10 12 14


A/2

B/2 Lesions / 104 DNA bases

The absorption of 3-photons by DNA bases can trigger photo-chemical reactions. The relaxation pathways involve base fragmentation and the further formation of covalent bonds between adjacent bases 42 . Formation of such photo-induced products mainly involves the pyrimidines (Thymine, Cytosine) through the production of cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone adducts (64 PP). Quantitatively speaking, direct interaction between 266 nm light (UVC) and DNA in aqueous solution predominantly yields the CPDs TT and TC and the 64 TT 43 . To determine the direct impact of the strong laser field on the irradiated DNA in aqueous solutions, we examined the formation of these photo-products, when aqueous DNA was irradiated by the 8x8 matrix of laser-induced LDP channels, which deposits ∼ 59 mJ.min−1 , in comparison to DNA exposed to 266 nm fs laser pulses under similar conditions (see SI appendix for details), which deposits ∼ 1.9 mJ.min−1 (Table 1). With direct UV irradiation, we detected the photoproducts in abundance (Figure S7). The presence of these same photo-products was negligible in the case of laser-induced LDP, especially in comparison to the yield of the indirect radical-mediated DNA bases modifications (Table 2). While previous studies of the effects of 800 nm fs laser-induced LDP in aqueous DNA assumed the absence of such photo-induced products 28,39 , we show here that the direct interaction between the strong laser field and diluted DNA in aqueous solution occurs, but remains marginal. LC-MS/MS analyses allowed us to discriminate most of the common radical-mediated chemical DNA base modifications 44,45 and to quantify their yields of production. The slopes extracted from the linear regression of the production kinetics as a function of the energy deposited in aqueous solution by laser and gamma irradiation give the yield of base modifications (Figure 2, Table 2, see SI appendix for details). Here, we consider separately two types of base modifications in DNA : i) the products of the radical-mediated oxidation of DNA, mainly by the hydroxyl radicals • OH; ii) the products of the radical-mediated reduction of DNA molecules, e.g. by the hydrated electrons e− aq . Overall, we observe that the total yield of products induced by the gamma irradiation is more than one order of magnitude higher than the yield of products induced by the LDP. We attribute such a low production yield, relative to that of sparse ionizing gamma radiation, to

UV fs laser 236±15 145±12 19±2


Photo-products Cyclobutane pyrimidine dimer TT Cyclobutane pyrimidine dimer TC (6-4) adduct 64 TT


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25 20 15 10 5 0 0

2

4

6


Figure 2: Production kinetics of oxidative damages to bases of ct-DNA as a function of the energy deposited in the irradiated aqueous solutions by: A. gamma-rays; B. the matrix of 8x8 laser-induced LDP channels. 1. dC products: U-Gly (red), Hyd-U (blue); dT products: T-Gly (purple), 5-FoU (cyan); 8-oxoG (green); 2. dC products: Imid-C (green), 5-OHC (blue), 5-OHU (cyan); dT products: Hyd-T (red), 5-HmU (purple). Each point represents the average and standard deviation of three independent experiments. The linear regression analyses are weighted by the standard deviation and give statistically significant evaluations of the yields of damages (see SI appendix for details). the high concentration of primary radicals generated by the LDP 40 . Laser filamentation confines energy deposition within a micrometric scale, into the close environment of plasma channels, where the generation of reactive radicals is strongly localized. Here, second order recombination between primary reactive species is greatly favored over their reaction with DNA molecules. The dense laser-induced ionization, which is characterized by a low yield of DNA base modifications, present common traits with high linear energy transfer ionizing radiations 46,47 . Table 2 also lists the relative yields of base modifications produced either by gamma and laser irradiation, i.e. the ratio between the yield of a specific modification and the yield of all the modifications analyzed under our experimental conditions. In aqueous solutions, the indirect effect mediated by the hydroxyl radicals


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Table 2: Yield of products in modifications per 104 DNA bases per Joule of deposited energy by γ-rays and laser-induced LDP channels in aqueous solutions of isolated DNA.

γ-rays

fs laser-induced LDP

15.3±0.1 | 5.7±0.04 % 18±1 | 6.7±0.4 % 6.4±0.4 | 2.4±0.1 % 25±2 | 9.3±0.7 % 6±1 | 2.2±0.4 %

1.05±0.01 0.79±0.01 0.29±0.03 1.71±0.01 0.16±0.01

35±2 | 13±0.7 % 4.0±0.3 | 1.5±0.1 % 22±1 | 8.1±0.4 % 74±5 | 27±2 % 64±10 | 24±4 %

2.41±0.02 | 18.4±0.2 % 0.17±0.01 | 1.3±0.1 % 0.75±0.05 | 5.7±0.4 % 2.9±0.2 | 22±2 % 2.83±0.07 | 21.6±0.5 %

0.01±0.001 | 0.00±0.00 % 0.07±0.01 | 0.03±0.00 %

0.002±0.0005 | 0.02±0.00 % 0.01±0.001 | 0.08±0.01 %

270±23 | 100±9 %

13.1±0.4 | 100±3 %

Oxidative Products

dC Products U-Gly Hyd-U Imid-C 5-OHC 5-OHU dT Products T-Gly Hyd-T 5-HmU 5-FoU 8-oxoG

| | | | |

8.0±0.1 % 6.0±0.1 % 2.2±0.2 % 13.0±0.1 % 1.2±0.1 %

Reductive Products

5,6-dHT Hydrate Total

is known to largely dominate the mechanism of DNA damage induced by the gamma irradiation 44,46–48 . The percentage of the oxidation products induced by both gamma or laser irradiation are relatively similar, which suggest that similar processes are involved in base modifications, i.e. mainly the oxidation by hydroxyl radicals. Especially noteworthy is that the yield of 8-oxoguanine did not overwhelm all the other oxidation products. As discussed previously, we examined the possibility of direct multi-photon interactions of the intense laser light with DNA molecules, which could yield DNA excitation and ionization. The formation of base radical cations via ionization of the guanine is favored because of its low ionization potential, relative to the other DNA bases 49 . Furthermore, even if ionization occurs on the other bases, the initial electron holes tend to relocate onto guanine, because of its lower oxidation potential, via electron transfer between bases along the polymer chain 50,51 . In aerated aqueous solutions, the hydration of guanine radical cations and further reaction with oxygen can yield 8-oxo-G 48 . Consequently, the direct ionization of DNA in aqueous solutions overwhelmingly leads to the formation of 8-oxo-G. Our measurement of the 8-oxo-G, which is quantitatively similar to other oxidatively-induced products (Table 2), confirms a negligible contribution of the direct effect on DNA molecules of the strong laser field within the framework of our experimental conditions. In-depth analyses of the LC-MS/MS results reveals subtle differences in the relative yield of radicalmediated DNA modifications induced either by gammarays or laser-induced LDP (Table 2). LDP generation yields an overproduction of U-Gly, 5-OHC and T-Gly, respectively +2.3 %, +3.7% and +5.4 %, compared to gamma irradiation. The addition of hydroxyl radicals to

pyrimidines (thymine and cytosine) leads to the production of C5 and C6 centered radicals, i.e. 5-hydroxy-5,6dihydropyrimin-6-yl radicals and to a lesser extent 6hydroxy-5,6-dihydropyrimin-5-yl radicals (Figure 3) 44 . In O2 saturated aqueous solutions, these radicals can rapidly react with oxygen to form hydroperoxyl radicals and then hydroperoxides (Figure 3.A).These hydroperoxides can subsequently decompose into a mixture of products, including 5,6-glycol products (U-Gly, 5-OHC and T-Gly) and products resulting from the fragmentation and rearrangement of the pyrimidine ring (U-Hyd, T-Hyd and Imid-C) 52,53 . In view of the high concentration of radicals species generated by the LDP, we propose that the second order reactions of hydroxyl radicals with intermediate C5 or C6-centered radicals result in the direct formation, and thus the increase, of 5,6-glycol products without any further intermediate steps (Figure 3.B). The high energy deposition per unit of volume involved in the plasma generation can be associated to a high dose rate effect, which is known to yield oxygen depletion 54,55 . The local high consumption of oxygen in the vicinity of LDP, in particular by reactions with hydrated electrons 56 , may result in a lower yield of products that arise from hydroperoxides. Because 5-FoU arises exclusively from the decomposition of an intermediate hydroperoxide 44,53 , i.e. 5hydroxyperoxymethyluracil (Figure 3A.b), the oxygen depletion may explain the notable underproduction of 5-FoU from the LDP generation, −5 % compared to the gamma irradiation. In addition, there is a significant laser-induced production of 5,6-dihydrothymidine (5,6-dHT) (Figure S8), which is usually only detected under hypoxic condition. Indeed, the absence of oxygen is conducive to the reaction of thymine with hydrated


The Journal of Physical Chemistry Letters A.

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B.

Figure 3: Proposed formation mechanisms of oxidative base modifications by the laser-induced LDP in DNA aqueous solution. See text for details. electrons, which gives thymine radical anions and upon protonation, 5,6-dihydrothyminyl-6(5)-yl radicals (Figure 4) 45,57 . The latter radicals can undergo further reduction to 5,6-dHT or oxidation to 5(6)-hydroxy-5,6dihydrothymine (Hydrate). Thus, the production of 5,6-dHT and relative increases in the yield of Hydrate (0.08 % versus 0.03 % for the gamma irradiation) are consistent with the depletion of oxygen due to the high concentration of laser-induced reactive radicals.

Figure 4: Proposed formation mechanisms of reductive base modifications by the laser-induced LDP in DNA aqueous solution. See text for details. Irradiation of Fricke solutions under different conditions allow us to highlight the oxygen depletion. We compare the Fe3+ production resulting from both laser and gamma irradiation, in O2 saturated versus aerated solutions (Figure S6). We show that the saturation of a solution with oxygen does not affect the yield of Fe3+ produced by the gamma-rays. Here, the sparse ionization guarantees that the concentration of reactive radicals is always negligible relative to the concentration of oxygen. The limiting factor in the Fe3+ production is the concentration of radiation-induced species. Conversely, the saturation of the solution with oxygen deeply affects the laser-induced production of Fe3+ . Here, high local concentrations of reactive radicals de-

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plete locally the oxygen. Oxygen depletion affects the efficiency of the chemical reactions underlying the Fe3+ production (Reactions 2 of the SI appendix). The initial local concentration of oxygen in LDP channels immediate environment becomes the limiting factor in Fe3+ production. Again, such observations are consistent with a source of high dose rate ionizing radiation 35,36,58 . We have conducted innovative and rigorous comparative chemical analyses of the DNA base modification mediated by either gamma-rays, fs laser-induced LDP or UV fs laser pulses in aqueous solutions, as functions of absolute energy deposition in water. From our observations, we resolved the second-order radical-mediated chemistry characteristic of laser-induced dense ionization and local depletion of oxygen indicative of high rate of energy deposition within the volume occupied by the plasma channels. Our results strengthen the paradigm that considers the laser-induced low-density plasma as a source of intense ionization, which exhibits common traits with sources of ultra high dose rate, high linear energy transfer ionizing radiation 29,30,40 . The present study suggests that the interaction of high intensity fs laser with biomolecules in an aqueous environment, above the threshold for ionization of water, can not be restricted to photo-chemical or thermomechanical effects. The effects mediated by the reactive radicals produced by ionization during fs laser-induced ablation 6,59,60 or even high intensity multi-photon microscopy in biological samples 61 , should not be ignored. In the present study, we do not regard the generation of these reactive species as drawbacks of the laser irradiation 62 . More generally we wish to convey the sentiment that the spatio-temporal resolution of fs laser beams coupled with wave front shaping techniques, potentially offers an unmatched control and precision over the generation of the reactive species produced upon the ionization of water in systems of biological interest. The achievement of such great control over the production of species directly involved in cell signaling, may pave the way for exciting new applications, e.g. the direct optical manipulation of cellular biological functions 63,64 . Such applications will benefit from a better understanding of the fundamental processes governing the generation of these laser-induced reactive species in aqueous environment and their interaction with biological compounds. Acknowledgement The authors would like to thank the Professor Jean-Paul Jay-Gerin, Andrew Bass, PaulLudovic Karsenti and Simon Lefebvre for assistance, helpful comments and suggestions. Financial support for this research was provided by the Canadian Institutes of Health Research, Grant no MOP 86676 and 81356, the Natural Sciences and Engineering Research Council of Canada, Grant no I2I 025301001, and the Canadian Institute for Photonics Innovations.


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Supporting Information Available The supporting information files are available free of charge. • The supporting information appendix (PDF) details several aspects addressed in the main manuscript: experimental procedures, numerical simulations, additional complementary results and analyses. • Web Enhanced Objects : – Movie S1: Independent control of the spatiotemporal distribution of the laser-induced plasma channels in water. – Movie S2: Control of the spatio-temporal distribution of the matrix 8x8 of laserinduced low-density plasma channels in water.

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DNA Base Modifications Mediated by Femtosecond Laser-Induced

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