Genetic damage

The femtosecond laser induced DNA relaxation and the subsequent genetic damage was firstly reported by Tsen et al in the laser irradiation experiment of the plasmid DNA from E. coli [11, 14]. This laser induced genetic damage was presumed to be the dominant detrimental factor that caused the reduction of bacterial viability. The

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argument was used to successfully explain the difference between the log-load reduction factor between the wild type and a strain that is deficient in DNA repairing protein [14]. Additionally, the reactive oxygen species produced by visible femtosecond laser was regarded as the dominant mechanism that cause the denaturation of DNA.

To obtain further insight of the femtosecond laser induced genetic damage, we have performed irradiation of various types of plasmid DNA by a visible femtosecond laser.

A general characteristic of all the plasmid DNA tested in this study is the laser power density threshold of the DNA conformation transformation. With the laser power density above certain threshold, the supercoiled structure would be relaxed to the linear, open circular form, or both of them. The interesting feature is, the supercoiled structure remain stable while the laser power density used is below the threshold, even the exposure time is prolonged to ensure laser fluence is equivalent (figure 25). The other common feature of the DNA relaxation is, that the large sized plasmid seems to be more sensitive to the visible femtosecond laser pulses and is easily to be relaxed comparing to the smaller plasmid with less base pairs ( figure 27 ).

Figure 27. Dependence between plasmid size and threshold power

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It is already demonstrated that ISRS could induce protein structure alteration through the disturbance of the weak links within protein molecules [13]. Analogously, the weak links exist in the nucleic acid structure could also be affected by the resonant vibration caused by ISRS and leads to the conformational change. A well know low frequency collective motion in DNA molecule in the range of 10–100 cm⁻¹, corresponding to the range of terahertz frequency (3×10¹¹ to 3×10¹² Hz) has been observed in several researches [5, 7, 38]. The visible femtosecond laser adapted in our study has significant spectral content at the frequency of the collective vibration and is able to bring the molecules into resonant oscillation. In principle, ISRS effect could result in an amplitude of the vibrational displacement in the range of of 0.01 to 1Å to a low frequency vibration mode in the condition of a moderate Raman scattering cross section and a reasonable excitation intensity [39].

In the quasi-continuity model of DNA, the two polynucleotide backbones are intertwining around the z axis and connected by the hydrogen bond spring between the complementary bases. Some of the hydrogen bonds would temporally open at thermal equilibrium. Every 20-50 intact hydrogen bond, there is an open bond that demarcate the DNA into individual segment [5]. When the molecule is brought into resonance, the vibration possess the feature of the coupled standing wave oscillators. As the coupling effect is present, the vibrational energy can be concentrated upon some of the segments at certain moments, and the effect will induce an amplified vibration amplitude within the segments. In a simplified model considering only 8 oscillators coupling proposed by Chuo et al., the amplified amplitude can increase 2.5 times than the case when no coupling is present [5]. In a realistic DNA molecule with far more oscillator segments, the amplification of the vibration becomes more significant, and such over concentrated vibration energy can lead to a quake like motion and a hole between polynucleotide

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chains that allows the intercalation of an external molecule or the start of DNA replication process.

According to our observation, the length of plasmid molecules is correlated to the power density threshold. The possible underlying mechanism is, that the larger the plasmid size is, the more intact segments are involved into the coupling resonant vibration of DNA, thereby increase the possibility to create gigantic vibration amplitude (quake) between the polynucleotide chains. When the amplitude exceeds the strain limit of associated chemical bonding, the conformation of DNA could therefore be affected.

For instance, in the case of the relaxation of supercoiled DNA into the open circular form, the resonant vibration deposit excessive energy than the strain limit of polynucleotide bond. One of the chain subsequently split off and release the elastic energy stored in the supercoiled structure, leading to the formation of the nicked circular DNA. If the vibrational energy is even concentrated onto certain segments via the coupling effect and impulse stimulation, the polynucleotide chain could break off and result in a DNA molecule with linear conformation.

An intriguing question about the DNA relaxation process is if the breakage is dependent on the genetic sequence or specialized mechanical structure of the plasmid DNA. As shown in the figure 28, the untreated, laser irradiated DNA, and the EcoRI digested untreated and laser irradiated pBS plasmid DNA were examined by EtBr stained agarose electrophoresis. According to the assignment of electrophoresis bands in section 4.7.2, we can identified the predominant band in the first lane was the supercoiled DNA from the untreated plasmid. The major band from the irradiated DNA in the lane 2 shown a less mobility than the untreated, which can be assigned as linear conformation relaxed by femtosecond laser according to the EcoRI predigested DNA shown in lane 4. In the third lane, the digested DNA shown a smear along the lane with several definite bands with relative strong intensity. The presence of those DNA

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fragments with shorter length suggests there could exist some weak points within the DNA chain that are easily detached when the vibrational energy is deposited to the molecules via ISRS. When the laser energy brings the molecules into coherent collective vibration, the antinodes of the vibrational modes are at where the maximum atomic displacement takes place. A possible explanation to the existence of the weak points is that the plasmid DNA could be cleaved at the antinodes when the molecules are excited via ISRS, and the excited plasmid DNA therefore break into small distinct fragments from the antinodes of the vibrational modes.

Figure 28. Examination on the specific cleavage site on pBS plasmid Image from EtBr stained gel electrophoresis containing untreated pBS plasmid (lane 1) ,the laser irradiated DNA (lane 2), the laser irradiated DNA digested by EcoRI (lane 3) and the untreated DNA digested by EcoRI (lane 4).

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Moreover, each plasmid DNA we tested in this study shows specified conformation transformation after laser exposure (Table 3) and the correlation between the resonant coupling mechanisms between the exact DNA structures is still elusive.

Table 3. Summary of the conformation transitions induced by the irradiation of femtosecond laser.

Our experimental result also demonstrated the influence of damping effect on the energy transfer via the resonant coupling. As demonstrated in the eq. (7), the maximum amplitude induced by ISRS effect will be reduced and hence lower the efficiency of energy transfer. Damping effect from the viscous solvent molecules could act as cushion that absorbs a part of the energy from the laser impulse. In a realistic cytoplasm, several dissipative effects coexist with DNA. Thus the genetic damage in a living cell induced by femtosecond laser could be reduced than the purified plasmid DNA investigated in this study.

To discuss the role that genetic damage plays in the E. coli inactivation, the experimental condition of the laser irradiation experiment should be carefully examined.

In a laboratory condition, the generation time of E. coli is about 15-20 min in a medium which is abundant in nutrition. In an environment that lacks of nutrition, the generation time could be as long as several hours, even one day. The bacteria used for our laser treatment is suspended in a nutritionless PBS buffer solution, and the treatment is taking place at the temperature of 20-22^。C. In such experimental conditions, the metabolism activity of the bacteria abates to a certain low degree, and the generation time will hence

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span to hours. As a consequence, it takes time that at least is longer than the lifespan of E. coli for the genetic replication errors to deposit effective reduction to cell viability.

Therefore, this detrimental factor will certainly influence the bacterial viability tested by the plaque assay but it has only minor effect on the metabolism activity during or right after the laser exposure. The respiratory assay, which was conducted 5 min after the end of laser irradiation, shown an instant cell activity impact of the femtosecond laser. The inhibited enzyme activities are unlikely correlated with the damaged DNA because of the instant commencement of the suppressed respiratory activity. Moreover, in some cases, the interaction time between the laser irradiation and bacteria is less than 15 min, which is even lower than the replication time in an optimal condition. Similarly, the changes of membrane structure, functions and mechanical properties are all detected while examining the bacteria cell fixed at the moment at the very end of laser irradiation.

Since the exposure time in these experiment is less than the replication time in our experimental condition, these alterations of the cell properties might be independent to the laser induced genetic damage. This inference clearly separates the contribution to the bactericidal effects of genetic damage from other detrimental factors caused from protein aggregation, such as cytoplasmic leakage and cell surface properties alteration.

Interestingly, the supercoiled plasmid DNA would not be relaxed by the laser with wavelength of 830 nm (figure 23(b)). In our experimental setup, the spectral width of laser pulse at 830 nm is about 20 nm, which has significant spectral component at the frequency of the collective vibration. However, no conformation transition is observed after the treatment with excessive laser fluence by using the infrared laser pulse. The observation did not violate our physical model because of the dependence of Raman scattering cross section on the excitation wavelength. In principle, the differential cross section varies inversely with the fourth power of the excitation wavelength.

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𝜎′ ∝ (𝜐₀− 𝜐_𝑣𝑖𝑏)⁴~ (9)

Where 𝜐₀ is the wavenumber of the incident radiation, and 𝜐_𝑣𝑖𝑏 is the wavenumber of the vibrational mode. In our case of low frequency vibration modes of biomolecules, the 𝜐_𝑣𝑖𝑏 is about 30 cm^-1 and the 𝜐₀ is 24096 cm^-1. The eq. (9) is dominant by the fourth power of the excitation wavelength because of the overwhelming contribution of the 𝜐₀term. As previously shown in eq. (8), the maximum amplitude excited by ISRS is proportional to the Raman cross section. Taking together, the ISRS resonant amplitude excited by 415nm laser could be 16 times than the one excited by 830nm laser. This significant difference in resonant oscillation amplitude could explain why the infrared femtosecond laser is not capable to induce significant conformation changes in both DNA and bacterial proteins (figure 15).

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Chapter 6 Conclusions

In this study, we have demonstrated that the femtosecond laser irradiation-induced E. coli inactivation is a combination effect of an early respiratory rate reduction and the later cell permeability, membrane structure and genetic damages resulted from the laser irradiation-induced protein inhibition via ISRS process. A schematic drawing describes the relationship between irradiation time and bacteria state is presented in figure 28. The proposed scenario is that a brief femtosecond laser irradiation directly inhibits the enzyme functions in the aerobic respiratory chain and causes an immediate oxygen consumption reduction within the irradiated bacteria. This compromised respiratory function may play a role in the early stage of bacteria inactivation by the femtosecond laser treatment. The effect is not only immediate but also can be activated with a very low laser power. In particular, the different respiratory enzymes show different susceptibility after the laser exposure, which would shed a light on the interaction mechanism of membrane protein between femtosecond lasers.

As the exposure time increases, the other detrimental factors such as genetic damages and the alteration of membrane surface properties will contribute to the inactivation and induce a high reduction of viability. Surface properties of bacteria changes after laser irradiation, whereby indicates the loss of some membrane function related to the cellular metabolism and the further obstruction of the energy production process. Moreover, the relaxation of supercoiled DNA indicate the occurrence of genetic errors during the cell replication stages.

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The future research focusing on how a brief femtosecond laser irradiation immediately affects the cellular respiratory system shall reveal the structure of proteins in the enzyme complex that are highly sensitive to the ISRS-mediated coherent molecular vibrations. The identification of the specific enzyme targets in respiratory chain of the femtosecond laser irradiation may contribute to the development of a new strategy to the pathogen elimination technology.

Figure 29

.

coli. The upper arrow denotes the time scale of the interaction and lower square specifies the activated events in the bacterial inactivation.

A possible application of this laser technology is to cure the bacterial infection by antibiotics-resistant strains. As stated in the above chapter, the maneuvering of the

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laser irradiation with the peak power density of 0.17 GW/cm2 and the exposure time of 10 min, the glucose dependent respiration could be suppressed to about 25% of the untreated cells. By using these parameters, the laser fluence deposited to each bacteria cell can thus be estimated as 30 J/cm2. This fluence is close and even less than the light fluence reported in the study of inactivation of Bacillus atrophaeus with blue light irradiation (470nm) and the treatment of Staphylococcus aureus with photodynamic therapy [40, 41].

A concept picture of the visible femtosecond laser treatment is schematically described in the figure 30. The laser is focused onto the infected area with appropriate optical parameter and scans around the tissue until the whole area is shined with enough fluence. Pathogenic microorganism in proximity of the irradiated area will lose certain degree of metabolism activity and lower its growth speed. Except for the bactericidal effect induced form the ultrafast light source, the photoexcitation of bacterial photosensitive porphyrins and bactericidal reactive oxygen species production could also be triggered simultaneously [40]. To summarize, the femtosecond laser treatment could be an effective strategy to overcome antibiotic-resistant bacteria infection and lower the usage of medicine and chemicals.

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Figure 30. A concept picture of the visible femtosecond laser treatment. The green and red arrows are the direction of scan of the laser focused spot.

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