Top Curr Chem (2015) 355: 1–32 DOI: 10.1007/128_2014_569 # Springer International Publishing Switzerland 2014 Published online: 8 November 2014
Photoinduced Processes in Nucleic Acids Mario Barbatti, Antonio Carlos Borin, and Susanne Ullrich
Abstract Photoinduced processes in nucleic acids are phenomena of fundamental interest in diverse fields, from prebiotic studies, through medical research on carcinogenesis, to the development of bioorganic photodevices. In this contribution we survey many aspects of the research across the boundaries. Starting from a historical background, where the main milestones are identified, we review the main findings of the physical-chemical research of photoinduced processes on several types of nucleic-acid fragments, from monomers to duplexes. We also discuss a number of different issues which are still under debate. Keywords DNA fragments Excited states Excitons Nucleobases UV radiation Contents 1 2 3 4
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Historical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Absorption and Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Photodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4.1 Monomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 4.2 Base Pairs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
M. Barbatti (*) Max-Planck-Institut fu¨r Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mu¨lheim an der Ruhr, Germany e-mail: [email protected] A.C. Borin (*) Institute of Chemistry, NAP-PhotoTech, The USP Consortium for Photochemical Technology, University of Sa˜o Paulo, Av. Prof. Lineu Prestes, 748 05508-000 Sa˜o Paulo, SP, Brazil e-mail: [email protected] S. Ullrich (*) Department of Physics and Astronomy, University of Georgia, Athens, GA 30602, USA e-mail: [email protected]
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4.3 Stacking, DNA Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Open Problems and Debates in the Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Deactivation of Adenine: Multiple Pictures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Cytosine: Tautomers, Triple Intersections, and Triplet States . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Why Does Thymine Have the Longest Lifetime? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Dynamics Simulation Cacophony . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 UVA or UVB: Which Is Most Dangerous? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Does Ultrafast Deactivation Matter for Photostability? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations (6–4)PD 1Me-Cyt 5F-Cyt 9Me-Ade ADC(2) Ade Ado AMD AMP AT CASSCF CC2 CI CPD Cyt d(ApG) dA dAMP DFT dT FU GC GP Gua IC MRCI R2PI T:T TC TT TA TDDFT
(6–4) Pyrimidine-pyrimidone dimer 1-Methyl-cytosine 5-Fluoro-cytosine 9-Methyl-adenine Algebraic diagrammatic construction to second order Adenine Adenosine Adiabatic molecular dynamics Adenosine monophosphate Adenine–thymine pair Complete active space self-consistent field Coupled-cluster to approximated second order Configuration interaction Cyclobutane pyrimidine dimer Cytosine Adenine–guanine dinucleotide Deoxyadenosine Deoxy-AMP Density functional theory Thymidine Fluorescence up-conversion Guanine–cytosine pair Gas phase Guanine Internal conversion Multireference configuration interaction Resonant two-photon ionization Stacked thymines Thymine–cytosine CPD Thymine–thymine CPD Transient absorption Time-dependent density functional theory
15 18 18 19 21 22 23 24 25 26
Photoinduced Processes in Nucleic Acids
TD-DFTB Thy TR-IY TR-PES TR-TAS TSH Ura W
3
Time-dependent density functional tight binding Thymine Time-resolved ion yield Time-resolved photoelectron spectroscopy Time-resolved transient absorption spectroscopy Trajectory surface hopping Uracil Water
1 Introduction Interest in photoinduced effects in nucleic acids is deeply linked to the history of DNA itself. In fact, less than one decade after Miescher isolated the “nuclein” in 1869 [1], the bactericidal effect of UV radiation was discovered and became a fertile field of investigation [2]. When it was finally determined in 1928 that this lethal effect of UV radiation was caused by its absorption by nucleic acids, it raised the first speculations that DNA could be related to growth and reproduction [3], a fact only finally confirmed in 1944 [4]. Since then, immense effort has been dedicated to understanding exactly how radiation and nucleic acids interact. One major motivation for this interest rests, naturally, on health aspects: UV radiation is the main environmental agent triggering skin carcinogenesis [5]. Furthermore, mutagenic effects of radiation have also helped to shape life on Earth. In fact, it might be that even the evolutionary selection of nucleic acids as hereditary agents was partially induced by their physical-chemical response to the very high levels of UV radiation in the early-biotic world. From these initial motivations, the research on photoinduced effects in nucleic acids also points to a future where optical devices will take advantage of the unique charge-transport properties of these biological polymers. In this chapter, we quickly survey many of these themes while pointing to specific literature. Most of these topics will also be examined in depth in the remaining chapters of this book. Given the overwhelming breath of the field, spreading over different disciplines, from basic physical chemistry to medical and applied engineering, even a quick survey needs to be limited to restricted boundaries. We will focus on experimental and theoretical research on physical-chemical aspects of how nucleic-acid fragments respond to photoactivation. This chapter is organized as follows. In Sect. 2 the main historical milestones of the field are summarized; a survey of the basic properties of the absorption and emission spectra of nucleic acids is presented in Sect. 3. In Sect. 4 the current state of knowledge concerning the photodynamics of several nucleic-acid fragments, from monomers to duplexes, is presented, based mainly on results from timeresolved experiments and computational modeling. Finally, in Sect. 5, several different topics currently under debate are discussed. Needless to say, the selection of these topics was a very personal choice, reflecting our specialties.
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2 Historical Background In this section we survey the main milestones in research on photoinduced effects in nucleic acids (Table 1), bringing us to the current state of this field. The knowledge that UV radiation may have a deleterious effect on organisms dates back to long before the discovery of how nucleic acids encode genetic information. In 1877, Downes and Blunt discovered that sunlight has a bactericidal effect [2] and later, in 1893, Ward demonstrated that this effect was caused by the “blue-violet half of the spectrum” [20]. At the dawn of the twentieth century, Finsen showed that the bactericidal action of UV radiation could be used to cure cutaneous tuberculosis [7]. For this discovery he was awarded the Nobel Prize in Physiology or Medicine in 1903. In the late 1920s, Altenburg demonstrated that UV radiation could induce mutations in fruit flies [3] and Gates found that the bactericidal action spectrum peaked at 260–270 nm and correlated with “some single essential substance in the cell” [21]. Gates also seems to have been the first to recognize that this substance could be nucleic acids. In fact, he noted that the association between nucleic-acid absorption and lethal action would have “wider significance in pointing to these substances as essential elements in growth and reproduction” [9], an exceptional insight which would be confirmed only years later when the role of DNA in heredity was finally determined [4]. Meanwhile, one can find, for instance, claims that nucleic acids “act as a protecting sheath to the encased proteins, thus preventing their dissociation by the action of ultraviolet” [22]. Table 1 Milestones in the investigations of UV effects on nucleic acids Year
Milestones
1865 1869 1877 1896 1903 1928 1928 1928 1944 1953 1962 1982 1995 2001 2002 2007
Maxwell theory of electromagnetism Miescher discovers DNA [1] Bactericidal effect of sunlight is discovered [2] Unna proposes that UV radiation could cause skin cancer [6] Finsen is awarded the Nobel Prize for using UV to treat cutaneous tuberculosis [7] Mutagenic effect of UV in fruit flies is observed [3] Relation between UV and skin cancer in animals is shown experimentally [8] Gates associates bactericidal action to nucleic acid absorption [9] Heredity function of DNA is discovered [4] Watson and Crick double-helix model is published [10] Photoinduced pyrimidine dimers as cause of DNA damage is proposed [11] UV-induced mutation hotspots in DNA are identified [12] Crystal structure of DNA photolyase from E. coli is determined [13] Time-resolved spectroscopy of nucleosides is published [14, 15] Conical intersections in nucleobases are computationally determined [16–18] Ab initio nonadiabatic dynamics simulations of cytosine–guanine pair are published [19]
Photoinduced Processes in Nucleic Acids
5
In 1896, Unna associated excessive exposure to solar radiation, especially UV, to skin cancer [6]. In the 1930s, through animal research, it was established that UV radiation could cause skin cancer [8, 23, 24]. Consequently, a large amount of effort was dedicated to the characterization of UV-induced carcinogenesis (see [25] for a review) and also photoinduced repair [26]. The determination of the molecular structure of DNA [10] paved the way for the discovery that photochemicallyformed pyrimidine dimers cause biological damage [11]. The spectroscopy of nucleic acids has been investigated since the 1930s [27– 30]. The very low luminescence of these compounds under physiological conditions – an important indicator of the occurrence of internal conversion – has also been known for a long time [29, 31, 32]. Nevertheless, time-resolved spectroscopy of nucleic acids has been a much more recent development [14, 15]. Computational investigations into the electronic excitation of nucleic-acid fragments date back to the 1970s, when a number of simulations based mainly on semiempirical approaches helped with the assignment of measured absorption spectra. These early works are reviewed in [33]. In the 1990s, with the advance of computational capabilities and theoretical methods, the first high-level simulations of the absorption and emission spectra of the nucleobases were reported [34– 36]. In the early 2000s, the importance of conical intersections for explaining the photophysics of nucleobases [16–18] emerged from a series of studies performed by a number of groups. A few years later, the first ab initio nonadiabatic dynamics simulations of nucleobases and base pairs were published [19, 37].
3 Absorption and Emission The five nucleobases composing DNA and RNA – adenine, guanine, cytosine, thymine, and uracil (Scheme 1) – are good UV chromophores, with absorption peaks at about 260 and 200 nm (Fig. 1 and Table 2) and maximum extinction coefficients between 8,000 and 15,000 M1 cm1 [28–30, 39, 42]. For a discussion of high resolution spectroscopy of nucleic acids, see de Vries [43]; for photoelectron spectroscopy, see DOI: 10.1007/128_2014_550. In comparison, the DNA spectrum peaks at 260 nm and has an extinction coefficient of about 6,800 M1 cm1 [38]. It presents strong hyperchromism and the absorbance of denatured DNA is about 30% higher than that of native DNA [44]. Together with urocanic acid (peaking at 280 nm) [45], proteins (peaking at 280 nm because of aromatic residues), and melanin (broad absorption below 300 nm) [46], DNA is one of the main UV chromophores in mammalian skin [47]. In fact, common analytical methods for determining the DNA concentration and protein contamination in DNA samples are based on the measurement of the 260-nm absorption and of the 280/260-nm absorption ratio [48]. Thanks to the UV absorption by the ozone layer in the atmosphere, the solar irradiance in this range of wavelengths is very much reduced at the Earth’s surface (Fig. 1) [40, 41]. Although this greatly decreases the probability of photoexcitation of DNA, it still occurs, being the main cause of skin cancer [49].
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Scheme 1 Natural nucleobases and base pairs
Fig. 1 Absorption spectra of native human DNA [38] and of DNA nucleobases in water [39]. Terrestrial [40] and extraterrestrial [41] solar UV irradiance
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Table 2 Band maximum (λmax), extinction coefficient (ε), and quantum yield (ϕ) for absorption and fluorescence of the nucleobases in water (W, pH 7) and in the gas phase (GP). sh - shoulder Base
Phase
Absorption λmax (nm)
Ade
W GP W GP W GP W W GP
260, 207 252, 207 252, 271 (sh) 293, 284 269, 230 290 (sh),~260 207, 269 258, 202 244, 205 (sh)
Gua Cyt Thy Ura
ε (103 M1 cm1)
Refs.
15, 12, 14, 10
[28, 39] [28] [39] [29] [39] [28] [30, 39] [42] [28]
10, 8 10, 8 8, 9
Fluorescence λmax (nm) ϕ (104)
Refs.
321
2.6
[32]
328
3.0
[32]
314
0.8
[32]
338 309
1.0 0.5
[33] [32]
DNA luminescence is very low, similar to its building blocks, whose quantum yields are on the order of 104 (Table 2) [32, 33]. This implies that dissipation of the UV radiation energy happens through internal conversion mechanisms, which time-resolved spectroscopy and computational simulations have determined to occur on picosecond timescales for individual nucleobases and paired bases, but which can take much longer in single and double strands [39].
4 Photodynamics Motivated by the need to understand the mutagenic and carcinogenic effects of UV radiation, photodynamic properties of the individual nucleobases, nucleosides, nucleotides, base pairs, and single and double strands have been intensively studied, as can be appreciated throughout this book. Collectively, the literature has demonstrated the validity of a bottom-up approach, in which the understanding acquired at the molecular level is transitioned, in a stepwise fashion, from minimal models to a realistic system. The individual steps contributing to this endeavor are reviewed below and a short survey of experimental results is given in Table 3. From monomers in the gas phase to DNA in vivo, different processes act to stabilize the excited nucleic-acid system (Fig. 2). At the monomer level (Fig. 2a), deactivation after UV excitation is ultrafast (subpicosecond to picosecond time scales) and involves conical intersections induced either by ring deformation or by hydrogen dissociation [85]. In the case of base pairs (Fig. 2b), the internal conversion pathways followed by the monomers are complemented by intermolecular ultrafast proton-transfer processes [86]. When dealing with stacked bases (Fig. 2c), single strands, and double strands, the situation is blurred, as the relaxation processes become very much dependent on the specific sequence of nucleobases. The overall picture is that monomer and base-pair pathways are replaced by – or may co-exist with – excimer processes [65]. In vivo, the situation is even more complex
W
D2O
(dT)18
GP W GP W W GP GP GP W GP W GP GP GP GP GP GP GP GP W
(dA)18
AT A2 T2 d(ApG)
Ura
1Me-Cyt Thy
5F-Cyt
Cyt
9Me-Ade Ado AMP Gua
Ade
100 6–10 (AG*)
τ3 (ps) – – – – – – – – – – – – – 0.1 – – – – – 0.124 (AG*/S0) 0.126 (AA*/S0) 0.1 (nπ*/S0)
τ4 (ns)
272
266
267 266 267 266 266 267 267 267 265 266 265 270 267 267 267 267 267 267 267 260
λpump (nm)
IR TA
[83]
[65]
[50] [53] [50] [53] [65] [50] [67] [50] [73] [74] [73] [74] [50] [59] [50] [59] [79] [79] [79] [80]
2 400 238 2 400 238 TA 2 400 2 400 2 400 TA 3 800 TA 3 800 2 400 3 800 2 400 3 800 3 800 3 800 3 800 TA/FU TA
Refs.
Method
[65]
[81, 82]
[51, 56, 59, 70, 75–78]
[51, 59, 62, 65, 70, 75–77]
[51, 56, 59, 62, 68–72]
[59, 62, 66]
[51–64]
Other Refs.
Table 3 Survey of experimental time constants for deactivation of nucleobases, derivatives, and other DNA fragments. When numbers are given for method, they indicate the probe wavelength (nm) for ionization in TRPES experiments
8 M. Barbatti et al.
W
d(AT)9.d(AT)9
TT ! T:T
W
(dA)18.(dT)18
Photoinduced Processes in Nucleic Acids Mario Barbatti, Antonio Carlos Borin, and Susanne Ullrich
Abstract Photoinduced processes in nucleic acids are phenomena of fundamental interest in diverse fields, from prebiotic studies, through medical research on carcinogenesis, to the development of bioorganic photodevices. In this contribution we survey many aspects of the research across the boundaries. Starting from a historical background, where the main milestones are identified, we review the main findings of the physical-chemical research of photoinduced processes on several types of nucleic-acid fragments, from monomers to duplexes. We also discuss a number of different issues which are still under debate. Keywords DNA fragments Excited states Excitons Nucleobases UV radiation Contents 1 2 3 4
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Historical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Absorption and Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Photodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4.1 Monomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 4.2 Base Pairs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
M. Barbatti (*) Max-Planck-Institut fu¨r Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mu¨lheim an der Ruhr, Germany e-mail: [email protected] A.C. Borin (*) Institute of Chemistry, NAP-PhotoTech, The USP Consortium for Photochemical Technology, University of Sa˜o Paulo, Av. Prof. Lineu Prestes, 748 05508-000 Sa˜o Paulo, SP, Brazil e-mail: [email protected] S. Ullrich (*) Department of Physics and Astronomy, University of Georgia, Athens, GA 30602, USA e-mail: [email protected]
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4.3 Stacking, DNA Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Open Problems and Debates in the Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Deactivation of Adenine: Multiple Pictures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Cytosine: Tautomers, Triple Intersections, and Triplet States . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Why Does Thymine Have the Longest Lifetime? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Dynamics Simulation Cacophony . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 UVA or UVB: Which Is Most Dangerous? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Does Ultrafast Deactivation Matter for Photostability? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations (6–4)PD 1Me-Cyt 5F-Cyt 9Me-Ade ADC(2) Ade Ado AMD AMP AT CASSCF CC2 CI CPD Cyt d(ApG) dA dAMP DFT dT FU GC GP Gua IC MRCI R2PI T:T TC TT TA TDDFT
(6–4) Pyrimidine-pyrimidone dimer 1-Methyl-cytosine 5-Fluoro-cytosine 9-Methyl-adenine Algebraic diagrammatic construction to second order Adenine Adenosine Adiabatic molecular dynamics Adenosine monophosphate Adenine–thymine pair Complete active space self-consistent field Coupled-cluster to approximated second order Configuration interaction Cyclobutane pyrimidine dimer Cytosine Adenine–guanine dinucleotide Deoxyadenosine Deoxy-AMP Density functional theory Thymidine Fluorescence up-conversion Guanine–cytosine pair Gas phase Guanine Internal conversion Multireference configuration interaction Resonant two-photon ionization Stacked thymines Thymine–cytosine CPD Thymine–thymine CPD Transient absorption Time-dependent density functional theory
15 18 18 19 21 22 23 24 25 26
Photoinduced Processes in Nucleic Acids
TD-DFTB Thy TR-IY TR-PES TR-TAS TSH Ura W
3
Time-dependent density functional tight binding Thymine Time-resolved ion yield Time-resolved photoelectron spectroscopy Time-resolved transient absorption spectroscopy Trajectory surface hopping Uracil Water
1 Introduction Interest in photoinduced effects in nucleic acids is deeply linked to the history of DNA itself. In fact, less than one decade after Miescher isolated the “nuclein” in 1869 [1], the bactericidal effect of UV radiation was discovered and became a fertile field of investigation [2]. When it was finally determined in 1928 that this lethal effect of UV radiation was caused by its absorption by nucleic acids, it raised the first speculations that DNA could be related to growth and reproduction [3], a fact only finally confirmed in 1944 [4]. Since then, immense effort has been dedicated to understanding exactly how radiation and nucleic acids interact. One major motivation for this interest rests, naturally, on health aspects: UV radiation is the main environmental agent triggering skin carcinogenesis [5]. Furthermore, mutagenic effects of radiation have also helped to shape life on Earth. In fact, it might be that even the evolutionary selection of nucleic acids as hereditary agents was partially induced by their physical-chemical response to the very high levels of UV radiation in the early-biotic world. From these initial motivations, the research on photoinduced effects in nucleic acids also points to a future where optical devices will take advantage of the unique charge-transport properties of these biological polymers. In this chapter, we quickly survey many of these themes while pointing to specific literature. Most of these topics will also be examined in depth in the remaining chapters of this book. Given the overwhelming breath of the field, spreading over different disciplines, from basic physical chemistry to medical and applied engineering, even a quick survey needs to be limited to restricted boundaries. We will focus on experimental and theoretical research on physical-chemical aspects of how nucleic-acid fragments respond to photoactivation. This chapter is organized as follows. In Sect. 2 the main historical milestones of the field are summarized; a survey of the basic properties of the absorption and emission spectra of nucleic acids is presented in Sect. 3. In Sect. 4 the current state of knowledge concerning the photodynamics of several nucleic-acid fragments, from monomers to duplexes, is presented, based mainly on results from timeresolved experiments and computational modeling. Finally, in Sect. 5, several different topics currently under debate are discussed. Needless to say, the selection of these topics was a very personal choice, reflecting our specialties.
4
M. Barbatti et al.
2 Historical Background In this section we survey the main milestones in research on photoinduced effects in nucleic acids (Table 1), bringing us to the current state of this field. The knowledge that UV radiation may have a deleterious effect on organisms dates back to long before the discovery of how nucleic acids encode genetic information. In 1877, Downes and Blunt discovered that sunlight has a bactericidal effect [2] and later, in 1893, Ward demonstrated that this effect was caused by the “blue-violet half of the spectrum” [20]. At the dawn of the twentieth century, Finsen showed that the bactericidal action of UV radiation could be used to cure cutaneous tuberculosis [7]. For this discovery he was awarded the Nobel Prize in Physiology or Medicine in 1903. In the late 1920s, Altenburg demonstrated that UV radiation could induce mutations in fruit flies [3] and Gates found that the bactericidal action spectrum peaked at 260–270 nm and correlated with “some single essential substance in the cell” [21]. Gates also seems to have been the first to recognize that this substance could be nucleic acids. In fact, he noted that the association between nucleic-acid absorption and lethal action would have “wider significance in pointing to these substances as essential elements in growth and reproduction” [9], an exceptional insight which would be confirmed only years later when the role of DNA in heredity was finally determined [4]. Meanwhile, one can find, for instance, claims that nucleic acids “act as a protecting sheath to the encased proteins, thus preventing their dissociation by the action of ultraviolet” [22]. Table 1 Milestones in the investigations of UV effects on nucleic acids Year
Milestones
1865 1869 1877 1896 1903 1928 1928 1928 1944 1953 1962 1982 1995 2001 2002 2007
Maxwell theory of electromagnetism Miescher discovers DNA [1] Bactericidal effect of sunlight is discovered [2] Unna proposes that UV radiation could cause skin cancer [6] Finsen is awarded the Nobel Prize for using UV to treat cutaneous tuberculosis [7] Mutagenic effect of UV in fruit flies is observed [3] Relation between UV and skin cancer in animals is shown experimentally [8] Gates associates bactericidal action to nucleic acid absorption [9] Heredity function of DNA is discovered [4] Watson and Crick double-helix model is published [10] Photoinduced pyrimidine dimers as cause of DNA damage is proposed [11] UV-induced mutation hotspots in DNA are identified [12] Crystal structure of DNA photolyase from E. coli is determined [13] Time-resolved spectroscopy of nucleosides is published [14, 15] Conical intersections in nucleobases are computationally determined [16–18] Ab initio nonadiabatic dynamics simulations of cytosine–guanine pair are published [19]
Photoinduced Processes in Nucleic Acids
5
In 1896, Unna associated excessive exposure to solar radiation, especially UV, to skin cancer [6]. In the 1930s, through animal research, it was established that UV radiation could cause skin cancer [8, 23, 24]. Consequently, a large amount of effort was dedicated to the characterization of UV-induced carcinogenesis (see [25] for a review) and also photoinduced repair [26]. The determination of the molecular structure of DNA [10] paved the way for the discovery that photochemicallyformed pyrimidine dimers cause biological damage [11]. The spectroscopy of nucleic acids has been investigated since the 1930s [27– 30]. The very low luminescence of these compounds under physiological conditions – an important indicator of the occurrence of internal conversion – has also been known for a long time [29, 31, 32]. Nevertheless, time-resolved spectroscopy of nucleic acids has been a much more recent development [14, 15]. Computational investigations into the electronic excitation of nucleic-acid fragments date back to the 1970s, when a number of simulations based mainly on semiempirical approaches helped with the assignment of measured absorption spectra. These early works are reviewed in [33]. In the 1990s, with the advance of computational capabilities and theoretical methods, the first high-level simulations of the absorption and emission spectra of the nucleobases were reported [34– 36]. In the early 2000s, the importance of conical intersections for explaining the photophysics of nucleobases [16–18] emerged from a series of studies performed by a number of groups. A few years later, the first ab initio nonadiabatic dynamics simulations of nucleobases and base pairs were published [19, 37].
3 Absorption and Emission The five nucleobases composing DNA and RNA – adenine, guanine, cytosine, thymine, and uracil (Scheme 1) – are good UV chromophores, with absorption peaks at about 260 and 200 nm (Fig. 1 and Table 2) and maximum extinction coefficients between 8,000 and 15,000 M1 cm1 [28–30, 39, 42]. For a discussion of high resolution spectroscopy of nucleic acids, see de Vries [43]; for photoelectron spectroscopy, see DOI: 10.1007/128_2014_550. In comparison, the DNA spectrum peaks at 260 nm and has an extinction coefficient of about 6,800 M1 cm1 [38]. It presents strong hyperchromism and the absorbance of denatured DNA is about 30% higher than that of native DNA [44]. Together with urocanic acid (peaking at 280 nm) [45], proteins (peaking at 280 nm because of aromatic residues), and melanin (broad absorption below 300 nm) [46], DNA is one of the main UV chromophores in mammalian skin [47]. In fact, common analytical methods for determining the DNA concentration and protein contamination in DNA samples are based on the measurement of the 260-nm absorption and of the 280/260-nm absorption ratio [48]. Thanks to the UV absorption by the ozone layer in the atmosphere, the solar irradiance in this range of wavelengths is very much reduced at the Earth’s surface (Fig. 1) [40, 41]. Although this greatly decreases the probability of photoexcitation of DNA, it still occurs, being the main cause of skin cancer [49].
6
M. Barbatti et al.
Scheme 1 Natural nucleobases and base pairs
Fig. 1 Absorption spectra of native human DNA [38] and of DNA nucleobases in water [39]. Terrestrial [40] and extraterrestrial [41] solar UV irradiance
Photoinduced Processes in Nucleic Acids
7
Table 2 Band maximum (λmax), extinction coefficient (ε), and quantum yield (ϕ) for absorption and fluorescence of the nucleobases in water (W, pH 7) and in the gas phase (GP). sh - shoulder Base
Phase
Absorption λmax (nm)
Ade
W GP W GP W GP W W GP
260, 207 252, 207 252, 271 (sh) 293, 284 269, 230 290 (sh),~260 207, 269 258, 202 244, 205 (sh)
Gua Cyt Thy Ura
ε (103 M1 cm1)
Refs.
15, 12, 14, 10
[28, 39] [28] [39] [29] [39] [28] [30, 39] [42] [28]
10, 8 10, 8 8, 9
Fluorescence λmax (nm) ϕ (104)
Refs.
321
2.6
[32]
328
3.0
[32]
314
0.8
[32]
338 309
1.0 0.5
[33] [32]
DNA luminescence is very low, similar to its building blocks, whose quantum yields are on the order of 104 (Table 2) [32, 33]. This implies that dissipation of the UV radiation energy happens through internal conversion mechanisms, which time-resolved spectroscopy and computational simulations have determined to occur on picosecond timescales for individual nucleobases and paired bases, but which can take much longer in single and double strands [39].
4 Photodynamics Motivated by the need to understand the mutagenic and carcinogenic effects of UV radiation, photodynamic properties of the individual nucleobases, nucleosides, nucleotides, base pairs, and single and double strands have been intensively studied, as can be appreciated throughout this book. Collectively, the literature has demonstrated the validity of a bottom-up approach, in which the understanding acquired at the molecular level is transitioned, in a stepwise fashion, from minimal models to a realistic system. The individual steps contributing to this endeavor are reviewed below and a short survey of experimental results is given in Table 3. From monomers in the gas phase to DNA in vivo, different processes act to stabilize the excited nucleic-acid system (Fig. 2). At the monomer level (Fig. 2a), deactivation after UV excitation is ultrafast (subpicosecond to picosecond time scales) and involves conical intersections induced either by ring deformation or by hydrogen dissociation [85]. In the case of base pairs (Fig. 2b), the internal conversion pathways followed by the monomers are complemented by intermolecular ultrafast proton-transfer processes [86]. When dealing with stacked bases (Fig. 2c), single strands, and double strands, the situation is blurred, as the relaxation processes become very much dependent on the specific sequence of nucleobases. The overall picture is that monomer and base-pair pathways are replaced by – or may co-exist with – excimer processes [65]. In vivo, the situation is even more complex
W
D2O
(dT)18
GP W GP W W GP GP GP W GP W GP GP GP GP GP GP GP GP W
(dA)18
AT A2 T2 d(ApG)
Ura
1Me-Cyt Thy
5F-Cyt
Cyt
9Me-Ade Ado AMP Gua
Ade
100 6–10 (AG*)
τ3 (ps) – – – – – – – – – – – – – 0.1 – – – – – 0.124 (AG*/S0) 0.126 (AA*/S0) 0.1 (nπ*/S0)
τ4 (ns)
272
266
267 266 267 266 266 267 267 267 265 266 265 270 267 267 267 267 267 267 267 260
λpump (nm)
IR TA
[83]
[65]
[50] [53] [50] [53] [65] [50] [67] [50] [73] [74] [73] [74] [50] [59] [50] [59] [79] [79] [79] [80]
2 400 238 2 400 238 TA 2 400 2 400 2 400 TA 3 800 TA 3 800 2 400 3 800 2 400 3 800 3 800 3 800 3 800 TA/FU TA
Refs.
Method
[65]
[81, 82]
[51, 56, 59, 70, 75–78]
[51, 59, 62, 65, 70, 75–77]
[51, 56, 59, 62, 68–72]
[59, 62, 66]
[51–64]
Other Refs.
Table 3 Survey of experimental time constants for deactivation of nucleobases, derivatives, and other DNA fragments. When numbers are given for method, they indicate the probe wavelength (nm) for ionization in TRPES experiments
8 M. Barbatti et al.
W
d(AT)9.d(AT)9
TT ! T:T
W
(dA)18.(dT)18
