J. Photochem.
Photobiol. B: Bid.,
I1 (1991)
269
269475
Two-quantum photoprocesses in DNA under picosecond laser W irradiation at 2 16 and 270 nm G. G. Gurzadyan,+ R. K. Ispiryau and K. Sh. Voskanyan 'Zazeragin Tekhnika” R & D Co., Shopron str.21, Yerevan, 375090 Armenia
(U.S.S.R.]
(Received January 29, 1991; accepted April 23, 1991)
K~oro!.s. link.
DNA, two-quantum photoprocesses, picosecond UV laser, cross-
Abstract It is demonstrated that at high power picosecond laser irradiation of 216 and 270 nm, two-quantum photodestructions of the DNA secondary structure, such as interstrand covalent crossIinks, “weak” crossbnks, and B+C conformational transition, take place. Thermal effects do not contribute to the observed effects.
1. Introduction At high power laser UV irradiation of DNA a two-step excitation of highlying electronic states of DNA bases takes place as a result of absorption of two light quanta via the intermediate singlet level S1 for picosecond excitation [ 1, 2 ] and the triplet level T1 for nanosecond excitation [ 3, 41. Most experiments have been carried out using the fourth harmonic of a Qswitched or mode-locked Nd:YAG laser with h = 266 run and a KrF excimer laser with A = 248 nm, which fits the maximum of the first absorption band of DNA. The energy of two-quantum excitation in this case (E = 9 - 10 eV) exceeds the ionization and dissociation limits of DNA nitrogen bases, and causes a photochemical reaction with high quantum yield [5-131. Induced two-quantum photoproducts differ from one-quantum low intensity products, but are similar to y-radiolysis products. It has been shown that high-power nanosecond and picosecond laser UV irradiation very effectively produces single-strand breaks in plasmid DNA [6, 7, 10, 111, single- and double-strand breaks in single- and double-stranded DNA [ 8, 12], strand breaks in poly(C) and poly(A) [ 131, interstrandcrosslinks in DNA [ 71,N-glycoside bond cleavage in nucleic acid components [4], RNA chain breaks [ 141, and RNA-protein crosslinks [ 14, 151. At the same time, at high intensities of irradiation the +Author to whom the correspondence should be addressed.
Elsevier Sequoia, Lausanne
270
quantum yield of one-quantum photoproducts (pyrimidine dimers) decreases owing to effective depopulation of the intermediate level Sr [ 16, 21. In the present study we investigated the formation of two-quantum photoproducts at A = 216 run irradiation which is on the edge of the second electronic absorption band. We discuss also the role of thermal effects upon picosecond laser UV irradiation. 2. Experimental
technique
and methods
of investigation
As a source of powerful UV laser radiation we employed a picosecond yttrium altinate:Nd laser with two amplifiers and frequency conversion into the fourth (A = 270 run) and fifth (A = 216 run) harmonics. The UV radiation parameters were as follows: energy of a single pulse E= 0.1-l mJ, pulse duration T= 15 ps, and repetition rate f = 2 Hz [ 171. Calf thymus DNA was received from the Molecular Physics Department of Yerevan State University, the molecular mass of the sample was lo7 Da. The purified DNA was dissolved in SSC buffer (0.2 M NaCl+ 0.02 M Na3CGH607.5.5Hz0, pH 7.0-7.5). Irradiation of aerated DNA solution (absorbance A = 1.O) was carried out in quartz cuvettes under continuous mixing. The photon density was varied with the help of a quartz lens by changing the distance between the lens and the cuvette. Energy measurements of radiant exposure were carried out by a thermocouple and a calibrated pyrodetector. The intramolecular crosslinks between strands of DNA were determined by monitoring the absorbance changes at 260 run during denaturation and subsequent renaturation of irradiated solutions. As is known, DNA molecules with one (or more) crosslink are completely renatured after heating up to 90-100 “C (denaturation) and subsequent cooling. The decrease in absorbance at 260 run after denaturation-renaturation permits an estimation of the quantity of DNA with crosslinks [ 181. The quantum yield of a photoreaction (both one- and two-quantum) is defined as the ratio of the number of molecules undergoing reaction to the number of absorbed quanta [7, 19, 201. This definition allows a direct comparison of the quantum efficiencies of one- and two-quantum reactions. Conformational changes in DNA were investigated by circular dichroism using a Roussel Jouan 2 CD spectrograph (Prance). The effect of powerful laser UV radiation on DNA melting temperature was investigated by ground state absorbance changes. Spectral measurements were carried out using a Specord M40 spectrophotometer. 3. Results and discussion As stated in Section 2, the quantity of DNA with crosslinks was determined by following changes in absorbance during denaturation and subsequent renaturation of irradiated DNA samples. However, we must note that base
271
destruction also causes changes in absorbance (a decrease) [ 1, 31. In order to take into account the contribution of base destruction to the changes in absorbance, we irradiated the denaturated DNA samples and defined the photochemical sensitivity AA/AH (Table l), similar to the method used for separate DNA bases [ 31. These results were used to correct the crosslinks’ quantum yield. This correction is only possible for the case of irradiation of denatured DNA samples. In the case of irradiation of native DNA no absorbance changes were observed: a decrease in optical density due to base photolysis is exactly compensated by its increase at the cost of the formation of local denatured sites. Figures 1 and 2 show the quantum yields of crosslinks in DNA at different wavelengths (216-270 nm) and different values of irradiance (data at 254 and 266 nm are obtained from ref. 7). In the range 10P5-lo7 W cm-a, the quantum yield of crosslinks is constant and does not depend on the fluence rate. Here we have one-quantum excitation. At values greater than lo7 W cm-’ the quantum yield of crosslinks becomes an order of magnitude greater, indicating the presence of two-quantum excitation. The excited molecules absorb a second quantum and as a result react more effectively. At high fluence rates (greater than 10’ W cmP2) the curves reach a plateau owing to saturation of one of the steps of the two-quantum excitation. TABLE
1
Photochemical
sensitivity
AA/AH of denatured DNA
/+(nm)
E, (W cm-‘)
AA/AH (cm* J-‘)
270 216 216
2x 107 9.6x IO6 5.3x lo7
2.26 0.46 0.53
H radiant exposure,
E irradiance.
IRRADIANCE , W/cm2 Fig. 1. Dependence of the quantum yield of crosslinks in DNA on the k-radiance of continuous wave 254 nm 17) (A), high power laser 266 nm [ 71 (Cl) and 270 nm (0) radiation.
272
1.5
v!
0 9 Y
1.0,
z g
0.5,
CY
0
1 107
tog IO8 IRRADIANCE , W/cm2
Fig. 2. Dependence of the quantum yield of cros.sIinks in DNA on the irradiance laser 216 nm radiation. TABLE
2
Values of melting temperature T, and temperature interval wavelength A, irradiance E and radiant exposure H
A
cm>
E (TV cm-a)
254 254 239 280 216 216 216 216 216
of high power
H (J cm-‘)
1o-3 10-a
z 10-3 z 10-a 1.7x107 3.1 x lo7 5.2 x 10’ 5.7x 10’ 9.6x lo7
0.1 0.8 0.15 2.8 0.04 0.04 0.1 0.1 0.1
T,
(“C)
62.4 80 72 67 Decrease Decrease 60.7 64.3 67.7 66.8 65.3
AT of DNA irradiated
at given
AT (“C)
Reference
11.7
This 21 21 21 22 22 This This This This This
Increase Increase Increase Increase 14.8 13.8 11.4 11.6 14.6
work
work work work work work
We have also investigated DNA melting when irradiated at 216 nm. Table 2 gives values of the melting temperature T,,the temperature interval AT, and the irradiation wavelength, h-radiance, and radiant exposure. We also give literature data for low intensity irradiation of DNA using a conventional mercury lamp [ 21, 221. It is obvious from the results in Table 2, that under low intensity UV irradiation (up to 1.7 X lo7 W cmU2) at all applied wavelengths (216, 239, 254, 280 run), the values of the DNA melting temperature are smaller than those of unirradiated samples, while the values of temperature interval are larger. These results are explained by a process of formation of local denatured sites in DNA upon UV irradiation. When increasing the intensity of laser UV radiation (216 run), both the temperature interval and melting temperature increase. Apparently, this is the result of “weak” interstrand crosslinks which collapse during heating, but are still stronger than the existing hydrogen bonds.
273
Fig. 3. Dichroism curves of non-irradiated DNA samples (l), irradiated at A=270 nm, E= 10’ W cmm2, H=1.2 J cm-’ (Z), h=216 nm, E=1.8X109 W cme2, H=0.08 J cmm2 (3) and A=216 nm, E=lO' W cm-‘, H=0.16 J cmm2 (4).
It must be emphasized, that the nature of the “weak” interstrand crosslinks is different from the above-mentioned “strong” interstrand crosslinks, and they need to be examined in detail in the future. Using the method of circular dichroism we investigated conformational changes in DNA. Dichroism curves of irradiated DNA are illustrated in Fig. 3. In cases (2) and (3), as well as at low intensity 270 nm irradiation, there are no obvious changes in the curves; in case (4) at 2 16 run and at comparably low intensities a difference in the “long-wave” peak of the curves is observed. This indicates the double spiral twisting and a B + C conformation transition. Transition between conformations B and C in the range of the cyclobutane dimer was assumed by Lang [23] on the basis of circular dichroism data. In our case a conformational transition B + C was observed at 216 nm (but not at 270 m-u). In this case the dirner output sharply decreases compared with irradiation at 270 run [24], while the contribution of other photoproducts increases. Thus, apparently the B+C transition is related to the existence of other photoproducts (non-dimers), in particular, inter-strand crosslinks. 4. The role of thermal effects in picosecond laser W DNA
excitation of
It was necessary to discover whether thermal effects take place upon laser irradiation of nucleic acids. Our recent results of non-linear thermal defocussing of 2 16 nm picosecond laser radiation in water due to two-photon absorption, permits determination of the variation in refractive index An and the changes in temperature AT in the irradiated part of the water. The experimental set-up and the interpretation of the results are described in detail in ref. 25.
274
At E=5 GW cm-2, the values An=10w5 and AT=0.12 “C are obtained. These results were found in pure water under two-photon absorption (50% absorbed energy). It must be noted that in the case of irradiation of DNA in an aqueous solution, with absorbance up to A= 0.4, the estimations for temperature variations will be similar to those in pure water. Thus, we can conclude that under high power picosecond laser UV irradiation of biomolecules in condensed medium at intensities up to several Gigawatts per centimetre squared, the thermal effects can be neglected. 6. Conclusion
It is revealed that under high power laser irradiation at 216 nm, the quantum yield of covalent interstrand crosslink formation increases by an order of magnitude. Crosslinks were shown to be formed by a two-quantum mechanism. Investigation of DNA melting shows that under high laser intensities at 216 run “weak” interstrand crosslinks are also formed, which are stronger than the hydrogen bond but still weaker than covalent crosslinks. At 216 run laser irradiation a B + C conformational transition of doublestranded DNA was detected. Thermal effects are shown to be of no importance for picosecond laser UV irradiation of DNA. Acknowledgment
We would like to thank Dr. S. G. Arutyunyan for helpful discussions. References 1 D. A. Angelov, G. G. Gurxadyan, P. G. Kryukov, V. S. Letokhov, D. N. Nikogosyan and A. A. Oraevsky, High-power ultrashort laser action on DNA and its components. In R. Hochstrasser, W. Kaiser and C. V. Shank (eds), Picosecond Phmwmma IZ, Springer, Berlin, 1980, pp. 336-339. 2 D. N. Nikogosyan and G. G. Gurzadyan, Two-quantum photoprocesses in DNA and RNA biopolimers under powerful picosecond laser UV irradiation, Laser Chem., 4 (1984) 297-303. 3 D. N. Nikogosyan, D. A. Angelov and A. A. Oraevsky, Determination of parameters of excited states of DNA and RNA bases by laser UV photolysis, Photochem. Photobid., 35 (1982) 627-635. 4 T. N. Menshonkova, N. A. Simukova, E. I. Budowsky and L. B. Rubin, The effect of high intensity ultraviolet irradlation on nucleic acids and their components, FEES I&t., 112 (1980) 299-301. 5 G. G. Gurzadyan, D. N. Nikogosyan, P. G. Kryukov, V. S. Letokhov, T. S. Balmukhanov, A. A. Belogurov and G. B. Zavilgelsky, Mechanism of high power picosecond laser UV inactivation of viruses and bacterial plasmids, Photo&em. Photobtil., 33 (1981) 835-838. 6 G. G. Gumadyan, D. N. Nlkogosyan, T. S. Balmukhanov and G. B. Zavilgelsky, The study of formation of single-strand breaks in the DNA chain under picosecond laser UV irradiation, Photobiochem. Photobiophgs., 4 (1982) 87-93. 7 G. B. Zavilgelsky, G. G. Gunadyan and D. N. Nikogosyan, Pyrimldine dimers, single-strand breaks and crosslinks induced in DNA by powerful laser W irradiation, Photobiochem. Photobiophgs., 8 (1984) 175-187.
275 8 J. Opitz and D. Schulte-Frohhnde,Laser induced photoionization and single-strand break formation for polynucleotides and single-stranded DNA in aqueous solution: model studies for the direct effect of high energy radiation on DNA, J. Photocti., 39 (1987) 145-163. 9 M. A. Chilbert, M. J. Peak, J. G. Peak, M. J. Pelhn, D. M. Gruen and G. A. Williams, Effects of intensity and fluence upon DNA single-strandbreaks induced by excimer laser radiation, Photo&em. Photobid., 47 (1988) 523-525. 10 D. T. Croke, W. Blau, C. OhUigin,J. M. Kelly and D. J. MCConnel,Photolysis of phosphodiester bond in plasmid DNA by high intensity W laser irradiation, Photo&em. Photobiol., 47 (1988) 527-536. 11 I. E. Kochevar and L. A. Buckley, Photochemistry of DNA using 193 run excimer laser radiation, Photo&em. Photobid., 51 (1990) 527-532. 12 E. Bothe, H. Giimer, J. Opitz, D. Schulte-Frohlinde,A. Siddiqi and M. Wala, Single- and double-strand break formation in double-stranded DNA upon nanosecond laser-induced photoionization, Photochern. Photobid., 52 (1990) 949-959. 13 M. Wala, E. Bothe, H. Giimer and D. Schulte-Frohlinde,Quantumyields for the generation of hydrated electrons and single-strandbreaks in poly(C), poly(A) and single-strandedDNA in aqueous solution on 20 ns laser excitation at 248 run, J. Photo&em Photobid. A: Chem., 53 (1990) 87-108. 14 E. N. Dobrov, Z. Kh. Arbieva, E. K. Timofeeva, R. 0. Esenahev, A. A. Oraevsky and D. N. Nikogosyan, W laser induced RNA-protein crosslinks and RNA chain breaks in tobacco mosaic virus RNA in situ, Photo&em. Photobiol., 49 (1989) 595-598. 15 E. I. Budowsky, M. S. Axentyeva, G. G. Abdurashidova, N. A. Simukova and L. B. Rubin, RNA-protein crosshnkin 30s subunitof Escherichia cob ribosome Induced by high intensity laser W irradiation, Dokl. Akad. Nuti SSSR, 281 (1985) 971-974 (in Russian). 16 D. N. Nikogosyan, G. G. Gurzadyanand G. B. Zavilgelsky, Pyrimidine dimer yield decrease in poly(dT) under high-intensitypicosecond laser W irradiation,Dokl. Akud. Nuuk SSSR, 269 (1983) 485-488 (in Russian). 17 A. G. Arutyunyan,.G. G. Gurzadyan and R. K. Ispiryan, Picosecond yttrium-aluminatelaser fifth harmonic generation, Kvantovaya Elektroniku (Sov. J. Quantum Electron.j, 16 (1989) 2493-2495. 18 G. B. Zavilgelsky, Kinetics of cross-links and local denatured sites induction in DNA under W irradiation. In G. M. Prank (ed.), Molec&r Biophysics, Nauka, Moscow, 1965, pp. 137-149. 19 D. N. Nikogosyan, Definition of photoreaction quantum yield at two-quantum excitation of molecules in solution, Laser Chem., 7 (1987) 29-34. 20 D. N. Nikogosyan, Two-quantum W photochemistry of nucleic acids: comparison with conventional low-intensity W photochemistry and radiation chemistry, Int. J. Rudiat. Bid., 57 (1990) 233-299. 21 G. B. Zavilgelsky and A. V. Zuev, Role of cyclobutane dimers in W-denaturation of DNA, Moleculyanuzya Biologiu, 12 (1978) 10661074 (in Russian). 22 R. B. Setlow and W. L. Carrier, Identification of ultraviolet-inducedthymine dimers in DNA by absorbance measurements,Photo&em. Photobiol., 2 (1963) 49-57. 23 H. Lang, CD studies of conformational-changesof DNA upon photosensitized W-Irradiation at 313 nm, Nucleic Acids Res., 2 (1975) 179-183. 24 G. G. Gurzadyan and R. K. Ispiryan, Efficiency of laser photolysis of nucleic acids at 216 run,Proc. Iti. Corlf. on Lasers in the Life Sciences, June 20-23,1990, Gruzng.zhou,China in the press. 25 G. G. Gmxadyan and R. K. Ispiryan, Nonlinear defocussing of laser radiation in nonlinear absorbing medium, J. Modern Opt., 38 (1991) 1265-1269.
Photobiol. B: Bid.,
I1 (1991)
269
269475
Two-quantum photoprocesses in DNA under picosecond laser W irradiation at 2 16 and 270 nm G. G. Gurzadyan,+ R. K. Ispiryau and K. Sh. Voskanyan 'Zazeragin Tekhnika” R & D Co., Shopron str.21, Yerevan, 375090 Armenia
(U.S.S.R.]
(Received January 29, 1991; accepted April 23, 1991)
K~oro!.s. link.
DNA, two-quantum photoprocesses, picosecond UV laser, cross-
Abstract It is demonstrated that at high power picosecond laser irradiation of 216 and 270 nm, two-quantum photodestructions of the DNA secondary structure, such as interstrand covalent crossIinks, “weak” crossbnks, and B+C conformational transition, take place. Thermal effects do not contribute to the observed effects.
1. Introduction At high power laser UV irradiation of DNA a two-step excitation of highlying electronic states of DNA bases takes place as a result of absorption of two light quanta via the intermediate singlet level S1 for picosecond excitation [ 1, 2 ] and the triplet level T1 for nanosecond excitation [ 3, 41. Most experiments have been carried out using the fourth harmonic of a Qswitched or mode-locked Nd:YAG laser with h = 266 run and a KrF excimer laser with A = 248 nm, which fits the maximum of the first absorption band of DNA. The energy of two-quantum excitation in this case (E = 9 - 10 eV) exceeds the ionization and dissociation limits of DNA nitrogen bases, and causes a photochemical reaction with high quantum yield [5-131. Induced two-quantum photoproducts differ from one-quantum low intensity products, but are similar to y-radiolysis products. It has been shown that high-power nanosecond and picosecond laser UV irradiation very effectively produces single-strand breaks in plasmid DNA [6, 7, 10, 111, single- and double-strand breaks in single- and double-stranded DNA [ 8, 12], strand breaks in poly(C) and poly(A) [ 131, interstrandcrosslinks in DNA [ 71,N-glycoside bond cleavage in nucleic acid components [4], RNA chain breaks [ 141, and RNA-protein crosslinks [ 14, 151. At the same time, at high intensities of irradiation the +Author to whom the correspondence should be addressed.
Elsevier Sequoia, Lausanne
270
quantum yield of one-quantum photoproducts (pyrimidine dimers) decreases owing to effective depopulation of the intermediate level Sr [ 16, 21. In the present study we investigated the formation of two-quantum photoproducts at A = 216 run irradiation which is on the edge of the second electronic absorption band. We discuss also the role of thermal effects upon picosecond laser UV irradiation. 2. Experimental
technique
and methods
of investigation
As a source of powerful UV laser radiation we employed a picosecond yttrium altinate:Nd laser with two amplifiers and frequency conversion into the fourth (A = 270 run) and fifth (A = 216 run) harmonics. The UV radiation parameters were as follows: energy of a single pulse E= 0.1-l mJ, pulse duration T= 15 ps, and repetition rate f = 2 Hz [ 171. Calf thymus DNA was received from the Molecular Physics Department of Yerevan State University, the molecular mass of the sample was lo7 Da. The purified DNA was dissolved in SSC buffer (0.2 M NaCl+ 0.02 M Na3CGH607.5.5Hz0, pH 7.0-7.5). Irradiation of aerated DNA solution (absorbance A = 1.O) was carried out in quartz cuvettes under continuous mixing. The photon density was varied with the help of a quartz lens by changing the distance between the lens and the cuvette. Energy measurements of radiant exposure were carried out by a thermocouple and a calibrated pyrodetector. The intramolecular crosslinks between strands of DNA were determined by monitoring the absorbance changes at 260 run during denaturation and subsequent renaturation of irradiated solutions. As is known, DNA molecules with one (or more) crosslink are completely renatured after heating up to 90-100 “C (denaturation) and subsequent cooling. The decrease in absorbance at 260 run after denaturation-renaturation permits an estimation of the quantity of DNA with crosslinks [ 181. The quantum yield of a photoreaction (both one- and two-quantum) is defined as the ratio of the number of molecules undergoing reaction to the number of absorbed quanta [7, 19, 201. This definition allows a direct comparison of the quantum efficiencies of one- and two-quantum reactions. Conformational changes in DNA were investigated by circular dichroism using a Roussel Jouan 2 CD spectrograph (Prance). The effect of powerful laser UV radiation on DNA melting temperature was investigated by ground state absorbance changes. Spectral measurements were carried out using a Specord M40 spectrophotometer. 3. Results and discussion As stated in Section 2, the quantity of DNA with crosslinks was determined by following changes in absorbance during denaturation and subsequent renaturation of irradiated DNA samples. However, we must note that base
271
destruction also causes changes in absorbance (a decrease) [ 1, 31. In order to take into account the contribution of base destruction to the changes in absorbance, we irradiated the denaturated DNA samples and defined the photochemical sensitivity AA/AH (Table l), similar to the method used for separate DNA bases [ 31. These results were used to correct the crosslinks’ quantum yield. This correction is only possible for the case of irradiation of denatured DNA samples. In the case of irradiation of native DNA no absorbance changes were observed: a decrease in optical density due to base photolysis is exactly compensated by its increase at the cost of the formation of local denatured sites. Figures 1 and 2 show the quantum yields of crosslinks in DNA at different wavelengths (216-270 nm) and different values of irradiance (data at 254 and 266 nm are obtained from ref. 7). In the range 10P5-lo7 W cm-a, the quantum yield of crosslinks is constant and does not depend on the fluence rate. Here we have one-quantum excitation. At values greater than lo7 W cm-’ the quantum yield of crosslinks becomes an order of magnitude greater, indicating the presence of two-quantum excitation. The excited molecules absorb a second quantum and as a result react more effectively. At high fluence rates (greater than 10’ W cmP2) the curves reach a plateau owing to saturation of one of the steps of the two-quantum excitation. TABLE
1
Photochemical
sensitivity
AA/AH of denatured DNA
/+(nm)
E, (W cm-‘)
AA/AH (cm* J-‘)
270 216 216
2x 107 9.6x IO6 5.3x lo7
2.26 0.46 0.53
H radiant exposure,
E irradiance.
IRRADIANCE , W/cm2 Fig. 1. Dependence of the quantum yield of crosslinks in DNA on the k-radiance of continuous wave 254 nm 17) (A), high power laser 266 nm [ 71 (Cl) and 270 nm (0) radiation.
272
1.5
v!
0 9 Y
1.0,
z g
0.5,
CY
0
1 107
tog IO8 IRRADIANCE , W/cm2
Fig. 2. Dependence of the quantum yield of cros.sIinks in DNA on the irradiance laser 216 nm radiation. TABLE
2
Values of melting temperature T, and temperature interval wavelength A, irradiance E and radiant exposure H
A
cm>
E (TV cm-a)
254 254 239 280 216 216 216 216 216
of high power
H (J cm-‘)
1o-3 10-a
z 10-3 z 10-a 1.7x107 3.1 x lo7 5.2 x 10’ 5.7x 10’ 9.6x lo7
0.1 0.8 0.15 2.8 0.04 0.04 0.1 0.1 0.1
T,
(“C)
62.4 80 72 67 Decrease Decrease 60.7 64.3 67.7 66.8 65.3
AT of DNA irradiated
at given
AT (“C)
Reference
11.7
This 21 21 21 22 22 This This This This This
Increase Increase Increase Increase 14.8 13.8 11.4 11.6 14.6
work
work work work work work
We have also investigated DNA melting when irradiated at 216 nm. Table 2 gives values of the melting temperature T,,the temperature interval AT, and the irradiation wavelength, h-radiance, and radiant exposure. We also give literature data for low intensity irradiation of DNA using a conventional mercury lamp [ 21, 221. It is obvious from the results in Table 2, that under low intensity UV irradiation (up to 1.7 X lo7 W cmU2) at all applied wavelengths (216, 239, 254, 280 run), the values of the DNA melting temperature are smaller than those of unirradiated samples, while the values of temperature interval are larger. These results are explained by a process of formation of local denatured sites in DNA upon UV irradiation. When increasing the intensity of laser UV radiation (216 run), both the temperature interval and melting temperature increase. Apparently, this is the result of “weak” interstrand crosslinks which collapse during heating, but are still stronger than the existing hydrogen bonds.
273
Fig. 3. Dichroism curves of non-irradiated DNA samples (l), irradiated at A=270 nm, E= 10’ W cmm2, H=1.2 J cm-’ (Z), h=216 nm, E=1.8X109 W cme2, H=0.08 J cmm2 (3) and A=216 nm, E=lO' W cm-‘, H=0.16 J cmm2 (4).
It must be emphasized, that the nature of the “weak” interstrand crosslinks is different from the above-mentioned “strong” interstrand crosslinks, and they need to be examined in detail in the future. Using the method of circular dichroism we investigated conformational changes in DNA. Dichroism curves of irradiated DNA are illustrated in Fig. 3. In cases (2) and (3), as well as at low intensity 270 nm irradiation, there are no obvious changes in the curves; in case (4) at 2 16 run and at comparably low intensities a difference in the “long-wave” peak of the curves is observed. This indicates the double spiral twisting and a B + C conformation transition. Transition between conformations B and C in the range of the cyclobutane dimer was assumed by Lang [23] on the basis of circular dichroism data. In our case a conformational transition B + C was observed at 216 nm (but not at 270 m-u). In this case the dirner output sharply decreases compared with irradiation at 270 run [24], while the contribution of other photoproducts increases. Thus, apparently the B+C transition is related to the existence of other photoproducts (non-dimers), in particular, inter-strand crosslinks. 4. The role of thermal effects in picosecond laser W DNA
excitation of
It was necessary to discover whether thermal effects take place upon laser irradiation of nucleic acids. Our recent results of non-linear thermal defocussing of 2 16 nm picosecond laser radiation in water due to two-photon absorption, permits determination of the variation in refractive index An and the changes in temperature AT in the irradiated part of the water. The experimental set-up and the interpretation of the results are described in detail in ref. 25.
274
At E=5 GW cm-2, the values An=10w5 and AT=0.12 “C are obtained. These results were found in pure water under two-photon absorption (50% absorbed energy). It must be noted that in the case of irradiation of DNA in an aqueous solution, with absorbance up to A= 0.4, the estimations for temperature variations will be similar to those in pure water. Thus, we can conclude that under high power picosecond laser UV irradiation of biomolecules in condensed medium at intensities up to several Gigawatts per centimetre squared, the thermal effects can be neglected. 6. Conclusion
It is revealed that under high power laser irradiation at 216 nm, the quantum yield of covalent interstrand crosslink formation increases by an order of magnitude. Crosslinks were shown to be formed by a two-quantum mechanism. Investigation of DNA melting shows that under high laser intensities at 216 run “weak” interstrand crosslinks are also formed, which are stronger than the hydrogen bond but still weaker than covalent crosslinks. At 216 run laser irradiation a B + C conformational transition of doublestranded DNA was detected. Thermal effects are shown to be of no importance for picosecond laser UV irradiation of DNA. Acknowledgment
We would like to thank Dr. S. G. Arutyunyan for helpful discussions. References 1 D. A. Angelov, G. G. Gurxadyan, P. G. Kryukov, V. S. Letokhov, D. N. Nikogosyan and A. A. Oraevsky, High-power ultrashort laser action on DNA and its components. In R. Hochstrasser, W. Kaiser and C. V. Shank (eds), Picosecond Phmwmma IZ, Springer, Berlin, 1980, pp. 336-339. 2 D. N. Nikogosyan and G. G. Gurzadyan, Two-quantum photoprocesses in DNA and RNA biopolimers under powerful picosecond laser UV irradiation, Laser Chem., 4 (1984) 297-303. 3 D. N. Nikogosyan, D. A. Angelov and A. A. Oraevsky, Determination of parameters of excited states of DNA and RNA bases by laser UV photolysis, Photochem. Photobid., 35 (1982) 627-635. 4 T. N. Menshonkova, N. A. Simukova, E. I. Budowsky and L. B. Rubin, The effect of high intensity ultraviolet irradlation on nucleic acids and their components, FEES I&t., 112 (1980) 299-301. 5 G. G. Gurzadyan, D. N. Nikogosyan, P. G. Kryukov, V. S. Letokhov, T. S. Balmukhanov, A. A. Belogurov and G. B. Zavilgelsky, Mechanism of high power picosecond laser UV inactivation of viruses and bacterial plasmids, Photo&em. Photobtil., 33 (1981) 835-838. 6 G. G. Gumadyan, D. N. Nlkogosyan, T. S. Balmukhanov and G. B. Zavilgelsky, The study of formation of single-strand breaks in the DNA chain under picosecond laser UV irradiation, Photobiochem. Photobiophgs., 4 (1982) 87-93. 7 G. B. Zavilgelsky, G. G. Gunadyan and D. N. Nikogosyan, Pyrimldine dimers, single-strand breaks and crosslinks induced in DNA by powerful laser W irradiation, Photobiochem. Photobiophgs., 8 (1984) 175-187.
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