Molecular Sciences
Literature Thesis
Photochemistry of viruses
by
Laura Finazzi
12907138
November 2020
12 ECTS
September 2020-November 2020
Supervisor/Examiner:
Examiner:
Abstract 5
Introduction 6
1 Viruses 8
1.1 Virus classification . . . 9
1.2 Virus life cycle . . . 11
2 Light mediated inactivation 15 2.1 Virus absorption of UV light . . . 17
2.2 Efficiency of disinfection . . . 19
2.2.1 Virus characteristics . . . 19
2.2.2 Radiation related factors . . . 20
2.3 Disinfecting UV wavelengths . . . 21
2.4 UV sources . . . 22
3 Photochemistry of virus inactivation 25 3.1 Deactivation mechanisms . . . 26
3.2 UV induced photoreactions . . . 29
3.2.1 Photoproducts of direct inactivation . . . 31
3.2.2 Photoproducts of indirect inactivation . . . 34
4 Photophysics fundamentals of nucleic acids 38 4.1 Isolated bases . . . 40
4.1.2 Cytosine and its derivatives . . . 42
4.1.3 Adenine and its derivatives . . . 43
4.1.4 Guanine and its derivatives . . . 44
4.2 Nucleic acid photophysics . . . 45
5 Experiments and applications 50 5.1 Advantages and disadvantages . . . 50
5.2 Experimental procedures . . . 51
5.2.1 Example of a kinetic model: the effect of temperature . 55 5.3 Applications . . . 56
6 Conclusions and future outlook 57
1.1 Enveloped and non-enveloped viruses . . . 10
1.2 Schematic representation of virus replication cycle . . . 12
2.1 UV section of electromagnetic spectrum . . . 17
2.2 Absorption spectra of nucleobases . . . 18
2.3 UV spectra of different UV lamps . . . 22
3.1 Photobiological, photochemical and photophysical processes time scale . . . 26
3.2 Virus deactivation mechanisms . . . 27
3.3 Virus life cycle disruption upon photodamage . . . 28
3.4 Photoproducts of direct UV deactivation . . . 33
3.5 Purine bases oxidation . . . 35
3.6 Pyrimidine bases type I reactions . . . 36
3.7 Oxidation of Guanine moiety . . . 37
4.1 PES of Uracil and Thymine . . . 42
4.2 Cytosine decay pathways . . . 43
4.3 Adenine decay pathways . . . 44
4.4 Guanine decay pathways . . . 45
4.5 Nucleobases and nucleotides assemblies . . . 46
4.6 Photophysical process in isolated unstacked DNA and stacked DNA . . . 47
4.1 Nucloside exciplex formation yields . . . 47 5.1 UV sensitivity of different viruses . . . 52 5.2 UV sensitivity of different bacteriophages . . . 53
Viruses are health relevant microorganisms, responsible for the transmis-sion of several diseases. The destruction and the prevention of their spreading is a major challenge in the worldwide public health.
Non-ionizing UV light affects living matter causing death of bacteria, mutation in animal cells, production of dangerous species and deactivating viruses. Thus, it is worthwhile to analyse the photochemistry of the latter components when hit by these wavelengths. Upon the absorption of UV radiation, viruses undergo molecular changes due to the presence of highly absorbing substances in their structure, such as proteins or nucleobases, re-sulting in an overall deactivation. Understanding the mechanisms of virus deactivation allows not only to expand the knowledge of the functioning of these pathogens, but also to exploit this interaction with UV light in differ-ent fields and applications, like in the bio-medical field to disinfect hospital rooms or samples.
In this thesis the photochemical mechanisms of virus deactivation are investigated and elucidated and the experimental conditions (e.g. dosage, wavelength, exposure time) required to deactivate viruses are reviewed. It is also highlighted how the overall photochemistry of viruses differs from their isolated building blocks, such as nucleobases. Understanding the structural fundamentals of virus functioning, reactivity and dynamics may facilitate the manipulation of particles in order to improve or develop new vaccines, gene therapy vectors, nanosystems for drug delivery or other biomedical or bio/nanotechnological ends.
Virology is increasingly capturing the attention of the whole scientific community, both because of the recent pandemic and because viruses provide outstanding and relatively simple models of biomolecular structure-function relationship linking physical, chemical and biological approaches. Acquiring knowledge on viruses functioning has a much deeper goal rather than the mere understanding on how to respond to viral diseases, it also unravels the physico-chemical mechanisms of molecular machinery of simple living organisms. Viruses are worth study for several reasons including:
• Virus particles are simple models to understand and manipulate molec-ular self-assembly.
• In order to understand all the phases of viruses life cycle, the virus structure, dynamics and properties still need to be fully elucidated. • Viruses are ideal models to explain structure-function relationships in
biomacromolecular assemblies and biological machines, as structurally they can be treated as nucleoproteins.
• Virus components and their biochemical reactions are the targets of the design of antiviral drugs.
• The structural basis responsible for virus stability, dynamics and func-tion constitutes a rafunc-tional model for the development of new or im-proved vaccines, gene therapy vectors, nanoparticles for drug delivery of other biomedical or bio/nanotechnological uses.
Viruses represent one of Nature’s models of efficiency. They are able to optimize their limited resources, due to their simple structure and the lack of metabolism, and they drive on a remarkable number of essential func-tions (genome protection, replication, transport, host cell infection). Despite the wide variety of fields studying the different biofeatures of viruses, the aspects related to their interaction with light have almost been overlooked until recently. Due to the spreading of SARS-CoV-2, the study of light me-diated virus inactivation has become a priority, not only in Academia, but also in the health care management. However, understanding the interaction between electromagnetic radiation and viruses could help not only to solve major worldwide health issues, but also to clarify biophysical mechanisms still unclear.
The aim of this thesis is to question the photostability of viruses and unravel the mechanisms and causes involved in viruses photodamage. This thesis will focus on how the photochemistry of nucleic acids, in particular their radiative and non radiative decay pathways, is related to virus deacti-vation.
The thesis is organized in the following way. The fundamental biofunc-tioning and properties of viruses are discussed, focusing on the replication mechanisms in Chapter 1. In Chapter 2, an introduction to light dis-infection is given and the factors affecting the efficiency of the process are explained; an overview of the main UV light sources and wavelengths com-monly used is given as well. In Chapter 3 the deactivation mechanisms in viruses are explained and the formation of photoproducts resulting from nucleic acid damage is described. In Chapter 4 the photophysics of the isolated nucleobases and nucleic acids is briefly explained. In Chapter 5 the advantages and drawbacks of this methodology are discussed, and ex-perimental procedures and applications of UV inactivation are presented. In the final chapter (Chapter 6) all the information are summarized and the research questions are answered.
Viruses
Viruses are biological submicroscopic intracellular parasites capable of as-sisted multiplication within cells and of propagation between cells and organ-isms. A virion is an infectious viral particle and, as it introduces its genome into a host cell, new virions are formed inside and released, and these sick cells may infect other host cells in a cyclic process [1]. Even though the terms virion and virus are commonly exchanged, they are not the same. According to Bandea’s definition, the virion is the infective particle whereas viruses is the infected cell [2]. Since virions are not able to undergo replication by themselves, viruses are not considered “autonomous” organisms as they are infectious parasites exploiting the resources of a host cell to carry out the pro-cesses needed for their propagation and replication. Furthermore, viruses do not possess any biochemical or physical potential, thus they cannot generate the energy required to assist all the biomolecular processes by themselves.
The cellular structure of virions is extremely simple as it lacks of the com-mon elements such as nucleus, cytoplasm, ribosomes and membrane. Their structure consists only in the genome, either one of the two nucleic acids, in some cases wrapped in an external protein coat. Almost all plant virus and some bacterial and animal virus have RNA genomes and these genomes tend to be particularly small. For instance, the genomes of mammalian retro-viruses such as HIV are about 9000 nucleotides long, and the genome of the
bacteriophage Qβ has 4220 nucleotides. Both viruses have single stranded RNA genomes. DNA viruses, on the other hand, have more size variety and many viral DNAs have a circular shape for at least a part of their life cycle. The different type of genomic acid inside different viruses is fundamental to determine the mechanisms of expression during the metabolically active phase of the replication cycle. The type of nucleic acid, and the distinction between single-stranded (ss) and double-stranded (ds) determines the assem-bly of the capsid, the mechanism of packaging, the organization of the nucleic acid inside the capsid, the maturation of the virus and properties related to the virion functions. An additional differentiation for single-stranded virus genome is between positive sense (+) (i.e. same polarity/nucleotide sequence as mRNA), negative sense (-) or ambisense (a mix of the two). Regardless the type and dimensions of the genome, an important requirment for the nucleic acid is that it must contain information encoded in such a way that it can be decoded by the host cell. Despite their small diameter (1 µm), the proteic shells of virions protect the genetic info contained in the nucleic acid genome from physical, mechanical, chemical or enzymatic damage. However, they are extremely elastic and capable to deform without breaking. Thus, the combination of these characteristics makes physical breaking of the virus very difficult. The capsid has also a fundamental role in the biochemistry of a virus as it initiates the infection by delivering the genome within the host cell. The type of virus determines the ease or difficulty of the delivery process.
1.1
Virus classification
There are several ways to classify viruses depending on their characteris-tics. Among the years, different classification systems have been established or altered as our perception of viruses changed. The main classification di-vides viruses into two large groups based on their molecular composition: non-enveloped and enveloped, depending on the absence or presence of an
outer lipid layer [3] (Figure 1.1). This outer shell consist in the repetition of one or more types of folded polypeptides subunits in a hollow symmet-ric protein oligomer or multimeter. The basic types of capsid symmetry are helical and icosahedral.
Figure 1.1: Different types of viruses. MVM and Φ29 are non-enveloped viruses. INFLUENZA is an enveloped virus. The scale is shown at the top left. [1].
The principal classifications used to group viruses are the following: • By disease: This classification is very straightforward and simple,
it suffers though from a lack of accuracy, as many diverse viruses can cause similar symptoms. For example, both influenza and SARS-CoV-2 cause fever and sore throat.
• By the host cell: Another simple way to classify viruses is to group them by the kind of host cell they are able to infect.
• By morphology: Due to the increasing knowledge and isolation of a large number of viruses, a classification based on the structure of virus particles is nowadays possible. Yet, it still is an issue to distin-guish among viruses that are morphologically similar but cause different clinical symptoms (e.g. the family of Picornaviruses). Nevertheless, morphology still continues to be an important aspect for virus classifi-cation.
• Functional classification: Identification of the replication strategy of the virus allows not only to classify the virus but also to understand how
the composition and structure of the virus genome and the constraints imposed on the replication mechanism are affected.
• Baltimore classification: In this system, viruses are placed into one of seven groups depending on the type of genome (DNA or RNA), strandedness (ss or ds), and method of replication.
1.2
Virus life cycle
The factors determining the replication stages of the virus are the nature of the viral genome and the type of host cell infected. For instance, RNA viruses do not need to penetrate the nucleus due to their structure, whereas DNA viruses replicate inside the nucleus, where the replication of the host cell takes place. However, some DNA viruses (e.g. Poxviruses) have developed the ability to replicate within the cytoplasm [4].
Virus replication happens in eight steps: attachment, penetration, un-coating, replication, assembly, maturation and release (figure 1.2). Every type of virus, regardless the nature of the host cell, must undergo all of these stages. However, describing a general replication process valid for every type of virus is extremely difficult since viruses can differ greatly from one to the other, and the different biochemistry of the possible host cells also needs to be considered. Nevertheless, the general virus replication takes place via the described stages and in this paragraph an overview is given.
The first stage, the attachment, consists in the binding between the virus through an antireceptor (a virus attachment protein) to the cellular receptor (a membrane protein or glycoprotein) of the host cell. This is the very first interaction between host cell and virus, thus it is a decisive step. Some viruses, like Poxviruses or Herpesviruses, have several routes to enter the cells as these viruses are able to use more than one receptor. In some cases interaction with multiple receptor is required to complete this stage. Although the virus attachment stage can differ greatly depending on the receptor, a common feature among receptors is that cells did not evolve to
allow viruses to penetrate in. The expression or absence of specific virus receptors on the host cell determines the tropism of a virus, i.e. the type of host cell the virus is able to infect.
Figure 1.2: Virus replication scheme [4].
The penetration step occurs shortly after the attachment of the virus to its receptor in the cell membrane. In contrast to the attachment, this process is energy-dependent. Thus, it is required that the host cell is metabolically active. In this phase the total or partial removal of the virus capsid takes place and the virus genome is ’poured’ into the host cell. The virus may enter the cell with three different mechanisms:
1. Translocation 2. Endocytosis
3. Fusion of the virus envelope with the cell membrane (only applicable to enveloped viruses)
Translocation is very unlikely to happen and yet to be utterly under-stood. It is mediated by capsid proteins and specific membrane receptors. Endocytosis is the most common mechanisms as it only requires normal func-tioning of the cell membrane. Fusion of the virus envelope can either follow
the endocytosis or happen directly at the cell surface. However, the former option requires the presence of a specific fusion protein in the virus envelope. After blending together the cellular and virus membranes, the nucleocapsid is dumped inside the cell into the cytoplasm.
Generally, the term uncoating is used to refer to all the processes taking place after penetration and its dynamics are still unclear. For instance, the removal of a virus envelope is considered part of the uncoating. This stage starts within the endosomes, probably triggered by pH changes, and then continues in the nuclear pores. The structure of the virus nucleocapsid is responsible for the uncoating products, as it determines the intermediate reaction steps.
The replication stage depends on the nature of the genome and, ac-cordingly to Baltimore’s classification, there are seven different ways it can take place depending whether the virus is single- or double-stranded, and its nucleic acid is RNA or DNA. This step is extremely important as it can determine the course of a virus infection (acute, chronic, persistent or latent). The structure of a virus particle is built in the assembly step. The aggregation of all the necessary components for the formation of the final product begins in a specific spot of the cell. The site of assembly directly depends on the replication site and the release mechanism of the virus from the host cell. For instance, Picornaviruses assembly within the cytoplasm whereas Polyomaviruses assembly in the nucleus. The site of assembly has also consequences on the maturation and release stages, and sometimes these three stages are coupled to each other.
Maturation is one of the final stages where the virus becomes infec-tious. Cleavages of the capsid proteins or internal alterations cause structural changes in the virus. These modification might lead to structural changes in the capsid, thus forming incomplete virus particle or inhibiting the process.
The last phase is the release of the virus particle and depends on the presence of the capsid. In non-enveloped viruses the process occurs easily as the infected host cell opens up and releases the virus. In enveloped viruses the
release happens after the formation of the virus lipid membrane, as the virus buds out of the cell through the cellular membrane or a vesicle. This process is called budding and its timing differs for each virus, as the membrane from which the virus buds depends on the type of virus involved.
Light mediated inactivation
The effect of light on pathogens can be dated back in 1878, when Arthur Downing found out experimentally how light could impact the development of bacteria [5]. Viruses can be inactivated by electromagnetic radiation and, depending on the nature of the light source, different mechanisms will occur. A basic distinction can be made between ionizing light and non-ionizing light. Ionizing radiation provides much higher photon energy than non ionizing, but it shows little selectivity and its physico-chemical effect on the virus is more governed by the atomic density of the virion [6].
Ionizing radiation When hit by ionizing radiation, the deactivation mech-anism can either be direct or indirect, as reactive species may be produced. For instance, if free radicals are released, a radical attack on the phage tail can cause a release of DNA from the phage head [7]. On the other hand, the direct inactivation involves the rupture of covalent bonds in a polypeptide or a nucleic acid. In the latter case, strand scission is more serious for single stranded nucleic acids than for double-stranded nucleic acids as parting com-plementary strands is required to disrupt double helices structures, whereas single-stranded nucleic acids can be broken anywhere. In fact, X-rays are ten times more efficient in deactivating viruses which contain single-stranded nucleic acids rather than those containing double-stranded nucleic acids.
Non-ionizing radiation Around 1930, the physician Frederick L. Gates became aware of the bactericidal action of ultraviolet (UV) light and mea-sured the wavelength dependence of the effect. In fact, in his work he stated that:
The close reciprocal correspondence between the curve of bacte-ricidal action and the curves of absorption of UV energy by these nuclear derivatives not only promotes the probability that a sin-gle reaction is involved in the lethal action of UV light, but has a wider significance in pointing to these substances as essential elements in growth and reproduction.
In opposition to ionizing radiation, non-ionizing ultraviolet light inacti-vates viruses without the rupture of proteins’ covalent bonds, as purine and pyrimidine rings of nucleic acids strongly absorb UV light. Proteins also ab-sorb these wavelength but more weakly and mainly because of the aromatic moieties but the deactivating effect is assumed to take place within the viral nucleic acid. Virus inactivation by UV light can be due to one or more of the following effects: new covalent bonds formed between adjacent thymine residues in DNA or between uracil residues in RNA to form so-called thymine dimers or uracil dimers, respectively; hydration at the C5-C6 double bond of pyrimidines to form 5-hydro-6-hydroxy derivatives (it is believed that these molecule have a primary role in the inactivation of RNA-containing viruses); cross-linking between complementary chains of double-stranded nucleic acids, probably through pyrimidines [8]. Furthermore, the photoinactivation of viruses results to be driven by molecular changes in nucleic acids due to the absorption of UV wavelengths, this is proved by the action spectra of pho-toinactivation having a shape similar to the absorption spectra of nucleic acids. Thus, the nucleic acids within the virus particle play a crucial role in the absorption of UV radiation and in virus inactivation. In most viruses the other major constituents of the virus particles play relatively minor roles in inactivation by UV [9, 10]. The photoinduced effects of UV radiation on
nucleic acids have widely risen interest not only in the biomedical research, in relation to health aspects such as cytodeath and carcinogenesis, but also in the prebiotic chemistry field. In fact, it could be possible that evolution drove nucleic acids to become life’s main building blocks due to their response to UV radiations. Future research on the photoinduced effects aims to project optical devices exploiting their charge-transport properties [11]. Besides the medical, chemical and evolutionary relevance, the photoproperties of nucleic acids have also shown potential for applications in material science [12].
2.1
Virus absorption of UV light
The disinfecting effect of ultraviolet light is widely known and accepted. UV radiation from the sun is a primary germicide in Nature. The UV part of the electromagnetic spectrum is divided in three parts differing by their wavelength: UV-C: 100-280 nm, UV-B: 280-315 nm and UV-A: 315-380 nm (fig. 2.1). The vacuum ultraviolet portion (VUV) is not considered for disin-fection purposes because it is strongly absorbed by air. Different wavelengths affect different chemical bonds in molecules. In fact, recently UV-C light has been found to be the most effective radiation for the destruction of DNA and RNA inside bacteria, viruses and protozoa [9, 13, 14]. On the other hand, UVA light does not have any germicidal effect whatsoever.
Figure 2.1: Electromagnetic spectrum with details in UV range. Source: LUX review
The chromophores responsible for the photodamage are proteins and ni-trogenous bases present within DNA and RNA. The nini-trogenous bases exhibit
strong absorption bands peaking in the range of 240-270 nm, due to the ππ∗
transitions of the purine and pyrimidine rings [15, 16]. In figure 2.2, the absorption spectra of nucleobases are shown. All the compounds display an absorption band at 200 nm and another more intense band at 260 nm. The latter is usually shifted to the blue for the pyrimidine compounds. The strong bands in the spectra are 1π → π∗ transitions. Other possible transitions are 1n → π∗ but weaker, due to their forbidden character [17].
The maximum absorption peaks of proteins are usually seen between 275 and 280 nm and the amino acids responsible for these peaks are tryptophan (Trp), tyrosine (Tyr) and to a small extent phenylalanine (Phe) and cysteine (Cys). The absorbances of these amino acids depend on the polarity of the environment: when polarity increases a blue-shift is observed.
2.2
Efficiency of disinfection
The factors affecting the efficiency of UV disinfection can be summarized in two main categories. These factors are discussed in more detail below.
• Virus characteristics: Factors related to the physiological state (pre-culturing, growth phase), strain diversity, repair mechanisms and par-ticle association.
• UV dose assessment: UV intensity and dose, absorption, reflection and refraction of UV light through the medium and lamp intensity, also affected by aging and fouling.
2.2.1
Virus characteristics
The physiological state can affect the sensitivity to environmental stress factors (e.g. UV radiation). The UV sensitivity is also related to the life stage of the virus and different strains of the same species may have different UV sensitivity. For instance, viruses containing single-stranded nucleic acids are more sensitive to UV damage, additionally, virus sensitivity also depends on the number of bases (thus on the size) present in the nucleic acid. In fact, virus sensitivity increases proportionally to the genome size, since the probability of hitting a larger target is higher at a given level of UV expo-sure. Another fundamental distinction in sensitivity between RNA and DNA nucleic acids is the formation of different photoproducts: pyrimidine dimers, in particular thymine dimers. Since DNA but not RNA contains thymine, DNA-containing viruses are generally more sensitive to damage by UV than RNA-containing viruses. Moreover, repair can reduce the lethal effect of UV, especially for viruses possessing double-stranded (ds) nucleic acids. Further-more double-stranded viruses sometimes present repairing mechanisms that reduce the lethal effect of UV light [9].
Repair mechanisms should also be considered as the UV radiation can damage not only the nucleic acids, but also other components of the virion
particle. However, even though viruses are not able to repair damage in-flicted to their genome by themselves because they have no metabolism, some viruses are able to benefit from the repair enzymes of the host cell. For instance Adenovirus, a double-stranded DNA virus, is able to exploit the host cell’s repair mechanisms, whereas RNA viruses may not [19].
2.2.2
Radiation related factors
The effectiveness of UV light disinfection is directly related to the UV dose absorbed by the targeted virus, since these systems respond linearly to photon flux. Hence, factors such as wavelength, UV intensity and UV dose influence the disinfection. In literature different terms can be found in the quantitative analysis of photochemical and photobiological processes. UV irradiance (W/cm2) and UV dose (J/cm2) are the most used
power-and energy-based units, respectively, but often UV dose is called “fluence”. Fluence is defined as “total radiant energy incident from all directions onto a small sphere divided by the cross-sectional area of that sphere” [20], but it is also referred to as UV dose. Since recent studies refer to UV dose as fluence, in this thesis will be done as well.
The role of the light source is crucial, as it determines the overall efficiency of the disinfection system. In order to achieve the maximum efficiency, the wavelength of the light source needs to match the maximum absorption of the chromophores present within the virus. The UV source must meet certain standards, performance criteria and guidelines. The optical characteristics of the medium (generally water) can influence the measurements to a great extent. The presence of UV absorbing organic and inorganic matter can reduce the UV fluence. Additionally, reflection caused by the construction materials of the UV reactor can influence the inactivation efficiency. The magnitude of this effect is greater in single lamp systems than in multiple lamp systems due to the higher surface-volume ratio. In the following section the most common used light sources and the germicidal effects of different wavelength wavelengths are illustrated.
2.3
Disinfecting UV wavelengths
Several case studies claim that the wavelength range of 220-222 nm has proven to be as efficient as the conventional germicidal UV light (254 nm) [13, 21–23]. The great advantage of UV-C is that it does not cause any health issues associated with exposure to UV light, thus, expanding its applications to many other fields and samples. The reason is that far-UVC light has a range in biological materials of less than a few micrometers, therefore, it cannot reach living cells in the human body as its penetration depth is very limited. Not even the human outer dead-cell skin layer can be penetrated. Nevertheless, it can still kill viruses and bacteria, due to the small size of these pathogens [22]. Bacteria, for instance, are less than 1 µm in diameter, whilst the average human cell have a diameter from about 10 to 25 µm. Thus, UVC light can penetrate throughout typical pathogens but cannot penetrate significantly the outer perimeter of the cytoplasm of human cells. The opposite reasoning applies to light of higher wavelengths, as they can reach human cell nuclei without any attenuation. The drawback of using this wavelength range is that it is not possible to exploit sunlight as UVC light source because it gets completely absorbed by the atmosphere and never reaches the Earth surface. Thus, suitable UVC source designs are required for this kind of decontamination [24]. However, far-UVC light shows great potential to be used in the decontamination of occupied public spaces and to prevent the airborne transmission of pathogen, such as Coronaviruses. In fact, no cytotoxic or mutagenic species neither in vitro or in vivo are produced when exploiting this wavelengths.
The most common wavelength for germicidal light is 254 nm as it deacti-vates biomolecules by attacking the structure of nucleic acids. Nevertheless, the emission at 254 nm represents a human health hazard, as it can cause cataracts and skin cancer [21]. The range between 260-265 nm has also shown great results against influenza viruses. Its mechanisms of action is mainly through nucleic acid damage. The range between 275-278 nm is able to deactivate biomolecules by distressing their protein structures. In fact,
these wavelengths match the peak absorption of Tryptophan, Tyrosine and Cysteine. As the functioning of a protein is directly related to its structure, these structural changes result in the inactivation of the targeted pathogen.
2.4
UV sources
Low pressure mercury lamp
Historically, the most common used type of lamp is the low-pressure mer-cury lamp. Low pressure UV lamps contain mermer-cury gas at pressures around 10 torr and upon electrical stimulation, the gas emits UV light in a narrow range with peak emission at 254 nm, other wavelengths are produced as well as shown in figure 2.3.
Figure 2.3: Broad band UV spectrum of different pressure UV lamps compared to the germicidal effect for E. Coli [25].
Low pressure UV lamps are made of an envelope of quartz glass or other UV-transmittant glass, a pair of electrodes, a ballast to provide the proper lamp current and a mercury amalgam; the amalgam is usually an alloy of mercury with another element, like indium or gallium. An electric current passes between the electrodes and heats up the mercury vapour, which subse-quently stimulates the electronic transition and gives the desired UV emission
[25]. Usually, about 60% of the electrical input is converted to light and the efficiencies of this type of lamps tend to be about 30-31%, even though they depend on ambient operating conditions and type of ballast. The glass wall temperature, specifically at the coldest location, determines the pressure of the vapour within the lamp, thus it determines the total UV output. The UV output may be reduced by undesired cooling effects.
Far UVC lamp
It is a novel type of lamp, called excimer lamp with a peak emission of around 222 nm. It is mercury-free but has a much lower efficiency (about 8%) than low pressure lamps. In this type of lamps, a modulated electrical field is applied to a quartz glass body filled with Xe gas, the quartz goal is to serve as a dielectric barrier and to prevent the formation of plasma from short-circuiting the electrodes. Establishing the electrical potential between the two dielectrical barriers leads to the formation of an excited molecular complex, that does not have any stable ground state under normal condi-tions. This excited molecular complex is named excimer and decomposes in nanoseconds, losing its excitation energy in the form of photons at a pre-cise wavelength. The wavelength of emission depends on the composition of the gas mixture inside the dielectric barrier [26]. However, conventional UV mercury-based lamps have higher germicidal efficiency, with respect to electrical energy consumption, than excimer lamps. Hence, upgrading the latter to a large scale remain a subject of future improvements in its energy efficiency.
LEDs
Light-emitting diodes (LEDs) have been described as a promising light sources to use instead of lasers because of their appealing characteristics, such as: wider emission bands, smaller in size, light weighted, inexpensive, greater flexibility in terms of the irradiation time, short turn-on time, they require low voltage and are easy to operate. An emerging technology for virus
disinfection is UV light-emitting diodes (UV LEDs). However, UV LEDs exhibit narrow emission spectra and lower wavelength UV LED are prone to have a low output power [27]. Thus, even though they are definitely a promising tool, they require the testing of the efficiency for each pathogen, in order to establish the sensitivity of different viruses at different wavelengths. Microplasma
Microplasma UV irradiation is a novel technology that has several ad-vantages over other sources. These lamps possess cavities of few millimeters filled with KrCl and the electrons discharge time-varying voltages that upon interaction with the gas result in a monochromatic UV output with a peak at 222 nm, depending on the gas different wavelengths can be produced. Another advantage is that these lamps do not contain toxic substances like mercury. They are able to generate high power (115 mW) with small (25 cm2) and thin (<6 mm) lamp design. Their stabilization time (15 s) favours
higher energy efficiency, intermittent disinfection and longer durability. An interesting feature of these lamps is that they allow to damage not only the viral genome but also the protein capsid, thus allowing to deactivate a wider range of pathogens. However, their use in viral disinfection is not widely applied, probably due to the lack of studies employing this technology and their relatively high cost compared to other lamps [28].
Photochemistry of virus
inactivation
The dogma of photochemistry was stated in 1818 by Grotthus and Draper and stated that “only absorbed light can produce a chemical change”. There-fore, in order to initiate a photochemical reaction, a photon must by absorbed by a molecule producing an excited state where the species is at higher en-ergy. Subsequently, the next series of events depends on several factors such as the molecular structure, the nature of the electromagnetic radiation and the reaction conditions [29]. A photobiological process takes place over a long period of time and goes through different stages; firstly, the excitation processes occur very rapidly, subsequently, thermal and dark reactions (i.e. processes where light is not involved) take place, ending with slow biological level. The time scale of these events is shown in figure 3.1. Photochemical and photobiological processes are both carried on by photon absorption by chromophores. However, for a photochemical process the photon absorption occurs in solution within a single molecule and the progress is measured in terms of the decrease in concentration of the chromophore. On the other hand photobiological processes implicate the photon absorption by a chromophore contained in a highly organized structure and the progress is measured in terms of the inactivation of the cell (or virus) in regards to a biological
func-tion (e.g. replicafunc-tion) [20].
Figure 3.1: Time scales in photophysics, photochemistry and photobiology processes. The upper scale is the time in ordinary units, while the lower scale in logarithm of time. The lines in the picture represent the time scale on which an event can occur [29].
3.1
Deactivation mechanisms
Inactivation of microbial pathogens can take place through three different damaging pathways: direct endogenous damage, indirect endogenous dam-age, and indirect exogenous damage. Understanding which mechanism dom-inates the inactivation for a specific virus is extremely important to deter-mine the environmental factors (e.g. irradiance, virus medium) affecting the process. All these mechanisms are strictly wavelength dependent. The chro-mophores inside of viruses (nucleic acids and proteins) do not absorb light of longer wavelengths than UV, thus, endogenous mechanisms mainly rely on UV radiation. On the other hand, exogenous mechanisms can be initiated by longer wavelengths as there might be present external sensitizers absorbing light in the visible region [30].
Endogenous Direct Indirect Capsid protein damage Genome damage PPRI 1O 2, H2O2, CO3- • , O2- •, •OH, 3Sens Exogenous Indirect PPRI PPRI
Figure 3.2: Schematic representation of different inactivation mechanisms in viruses. The orange stars represent the site of damage (i.e. where the photon is absorbed by a chromophore). For direct mechanisms, the photon is absorbed by a chromphore at the site of damage. For indirect mechanisms, the photon is absorbed by a sensitizer and the damage occurs at a different site.
Direct damage is due the absorption of radiation by chromophores (such as nucleic acids or proteins) inside the virus, whilst indirect damage is the consequence of the attack to internal microbial components by photo-produced reactive intermediates (PPRI). PPRI include hydroxyl •OH, carbonate
rad-ical CO−•
3 , singlet oxygen1O2 and other reactive oxygen species (ROS) such
as superoxide O−•
2 and hydrogen peroxide H2O2, and triplet state organic
species. External chromophores that are responsible for the production of these reactive species are called sensitizers. Indirect damage may be either endogenous if it occurs within a constituent of the virus, or exogenous if the damaged species is not a constituent of the virus [10, 31]. Due to virus simple structure, indirect endogenous damage is often neglected. Exogenous damage is caused by the formation of PPRI by external sensitizers present in the virus medium. Even though, the three different mechanisms are sep-arately represented in figure 3.2, they probably occur simultaneously and synergistically with each other.
Both the endogenous or exogenous damage to viral nucleic acids can po-tentially end the virus life cycle. Depending on where the damage occurs,
ei-ther in the genome or in the capsid, the life cycle will be disrupted differently (Figure 3.3). Once the nucleic acid has been damaged and the photoproduct, which is usually a dimer, has formed, the virus particle is not able to repli-cate its genetic material anymore. However, there is still no literature on the impact of UV damage on the virus life cycle. Only two studies investigated the replicating steps inhibited by radiation [32, 33].
Figure 3.3: Stages of the virus life cycle upon damage. The phases in the picture are (i) attachment; (ii) entry; (iii) replication of nucleic acids and translation of proteins; (iv) assembly of virions and release by host cell [31].
Endogenous mechanisms Upon sunlight absorption, it has been proven that all viruses undergo endogenous inactivation. Nelson et al. mentioned in their review that, among the variety of viruses studied, the Human Ade-novirus (HAdV) and MS2 are the most resistant, whilst somatic phages and Poliovirus are more sensitive [31]. Generally, direct endogenous deactivation is more likely to happen rather than the indirect mechanism. The reason behind that lies in the elementary structure of viruses; as their biochemistry is limited and simple, there are not many internal photosensitizers matching the light source, thus, endogenous indirect inactivation is not likely to be an
efficient deactivation mechanism since it also takes place with a significantly slower rate. In addition to that, endogenous inactivation can damage both the protein capsids and the viral genome. Nevertheless, it still is a challenge to distinguish the two processes experimentally and the molecular mecha-nisms of these processes still need to be utterly understood [34]. Various case studies discovered that, depending on the type of virus, the inactivation could either depend on each genome lesion, such as MS2 [35], or only on one or more specific lesions, as in ssRNA Tobacco Mosaic Virus.
Recently, it has been discovered by Nishisaka-Nonaka et al. that UV radiation is able to inhibit host cell replication and transcription of viral RNA through suppressing the accumulation of intracellular mRNA (messen-ger RNA), vRNA (viral RNA), and cRNA (complementary RNA) [36]. Exogenous mechanisms When the virus medium (e.g. water) contains naturally occurring external sensitizers, they can influence the inactivation rates, speeding the overall process. The type of virus, together with the presence of exogenous sensitizers, is one of the factors affecting the rate of exogenous activation.
As mentioned in the previous sections, the production of PPRI species can affect the exogenous inactivation, even though it is extremely difficult to separate their effects from the endogenous inactivation. Yet, there is not sufficient literature on the damage of PPRI, besides 1O
2. However, it
has been found that 1O
2 can cause a chemical modification to the assembly
capability of the capsid protein.
3.2
UV induced photoreactions
Upon light absorption, a chromophore (C) absorbing a photon is pro-moted to an excited singlet state ( 1C*). This singlet excited state is short
lived (lifetime of nanoseconds) and generally returns to the ground state through non radiative (heat) or radiative processes (fluorescence). However,
the excited state may also go through intersystem crossing (ISC) to triplet ex-cited states (3C*), which have a longer lifetime (microseconds) [37, 38]. The
first law of photochemistry states that “only the light which is absorbed by a molecule can be effective in producing photochemical change in the molecule”, thus the excited species may either undergo photophysical or photochemical transformation, in the latter case the endogenous direct inactivation follows. Nucleic acids present in biomolecules are responsible for causing direct pho-toreactions, specifically adjacent pyrimidine nucleobases (cytosine, thymine and uracile) [8].
The structural and chemical properties of nucleic acid have huge impact on the photoreactivity. The base sequence determines the efficiency and dis-tribution of pyrimidine dimerization photoproducts; in fact, higher yields are observed for TT dimerization when flanking a pyrimidine versus a purine bases [39]. The photoreactivity is also affected by a large number of param-eters such as the ionic strength [40] and pH of the solution, as the rate of photolyses of the excited species is a function of pH [41].
Aside from direct photochemical reaction, the excited triplet species 3C*
may also induce sensitized processes, resulting in exogenous or endogenous indirect inactivation. The excited triplet species can act as an oxidant or photosensitizer and yield photoreactive intermediates, such as reactive oxy-gen species (ROS). ROS are biologically active species arising from oxidation of various organic compounds and they are capable of biocellular damage. ROS may also arise from mitochondrial respiration [42, 43]. The term ROS generally collects the first generation of species generated by oxygen reduc-tion (superoxide O•−
2 or hydrogen peroxide H2O2) and the secondary reactive
products.
The majority of reactive intermediates reacts with electron-rich sites con-tained in biomolecules. It has been proven, both theoretically [44] and exper-imentally [45], that pyrimidines are better electron acceptors than purines and guanine is the best π-electron donor among the four nucleic bases. For instance, guanine sites in nucleic acids are oxidized steadily, whilst in proteins
the electron rich amino acid side chains are preferred (tryptophan, tyrosine, histidine, methionine, cysteine and cystine).
Hydroxyl radicals react with all the amino acid side chains and backbones, but they preferentially react with arginine, lysine, proline and threonine, leading to carbonylic products. Furthermore, •OH hydroxylates aromatic
amino acids [31, 46].
3.2.1
Photoproducts of direct inactivation
The photochemical products of the absorption of UVB radiation on DNA are dimeric photoproducts [8, 47]. Cyclobutapyrimidines (Pyr<>Pyr) are the most abundant DNA lesion and they are formed via a [2+2] cycload-dition of the C5-C6 double bonds of adjacent pyrimidine bases [48]. The stereochemistry of the products depends on the position of the pyrimidine moieties, with respect to the cyclobutane ring (cis/trans stereochemistry) and on the orientation of the C5-C6 bonds (syn/anti regiochemistry); more-over, only syn isomers are generated due to steric strain and the cis-syn form is produced in large excess with respect to the trans-syn diastereomers [49]. Thymine dimers are mutagenic products able to disrupt the normal cellu-lar processing of DNA and lead to chain reactions of biological responses, including apoptosis, immume suppression and carcinogenesis.
A second class of photoproducts is pyrimidine(6-4) pyrimidone adducts, they arise from a [2+2] cycloaddition involving the C5-C6 double bond of the 5’-end pyrimidine and the C4 carbonyl group of the 3’-end thymine, yielding an unstable oxetane. This compound spontaneously rearrange into the adduct that upon exposure to UVB radiation gives the Dewar valence isomers.
The presence of a CC→TT mutation is another signal of UV photodam-age, however, they are present at 5-15% of mutation or sometimes not seen at all [50]. Although, it is still lacking enough documentation on the nat-ural origin of this mutation but an attempt to explain it suggests that CC photolesions have a high mutagenicity.
Irradiation of DNA also produces cytosine photohydrate. The formation of 6-hydroxy-5,6-dihydrocytosine occurs efficiently within monomeric model systems and it involves the nucleophilic addition of a water molecule to a reactive intermediate, likely a lowest-lying singlet state S1 or a vibrationally
excited state derived from S1. These photohydrates may also undergo
deam-ination to uracil hydrates, a more stable form. Although it is mainly pyrimi-dine bases that absorbing UVB radiation, purine bases can be photoreactive too. Adjacent adenine residues react together to form a dimer. Guanine moieties are oxidized upon exposure to UVC and UVB radiation without any photosensitizer [51]. A summary of all the photoproducts formed due to UV photodamge is shown in fig 3.4.
(a)
(b)
(c)
(d)
Figure 3.4: Photoproducts of UV radiation on polynucleotides [47] a: Formation of cyclobutane dimers and the different diastereoisomers ; b: Thymine (6-4) photoproduct formation and isomerization ;
c: Formation and deamination of cytosine hydrate;
3.2.2
Photoproducts of indirect inactivation
The two types of competitive mechanisms involved in the indirect deac-tivation are the type I (one-electron oxidation or hydrogen abstraction) and type II (singlet oxygen oxidation) mechanisms.
Type I photooxidation reactions
Generally, base moieties of DNA are the preferential substrate for the one-electron oxidation reaction. Guanine, due to its lowest ionization potential, is the preferred target, it is also able to undergo hole transfer from pyrimidine and adenine radical cations or lose an electron, thus, leading to the presence of a large amount of guanine radical cations. These charged species can undergo two competitive chemical processes: hydration and deprotonation. Purine bases oxidation The hydration of guanine radicals yields 8-oxo-7,8-dihydroguanyl radical, a reducing species. This radical undergoes re-duction forming 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). This reduction is in competition with oxidation, possibly achieved because of the presence of molecular oxygen, yielding 8-oxo-7,8-dihydroguanine (8-oxoGua). However, the deprotonation of the radical species produces 2,2,4-triamino-5(2H)-oxazolone through the formation of the instable imidazolone. The mechanism contains the addition of O2 and a H2O molecule to the
ox-idizing radical, previously obtained from the deprotonation of the guanine radical cation, followed by the rearrangement of the purine ring (figure 3.5).
Figure 3.5: Oxidation of purine bases of DNA; the guanine moiety is taken as example in these reactions [47].
Pyrimidine bases oxidation Pyrimidine bases are less prone to undergo one-electron oxidation than their purine analogues. Hydration at C-6 is also the main decomposition for thymine and cytosine. The obtained rad-ical reacts with molecular oxygen and undergoes reduction and protona-tion, yielding four diastereomers of cis- and trans-5-hydroperoxy-6-hydroxy-5,6-dihydropyrimidine derivatives of thymidine and 2’-deoxycytidine, respec-tively (Figure 3.6).
Figure 3.6: Type I reactions of pyrimidine type bases; in this case thymine moiety [47].
Type II photooxidation reactions
The reactivity of these kind of reactions is rooted in the formation of singlet molecular oxygen (1O
2) in its lower energy state ∆g. This highly
reactive species only reacts with electron-rich molecules, thus the ideal can-didate among the DNA bases will be guanine. The oxidation process in-volves a [4+2] Diels-Alder cycloaddition yielding unstable diastereomeric
en-doperoxides. Subsequently, these compounds decompose into 4-hydroxy-8-oxo-4,8-dihydro-2’-deoxyguanosine and may further rearrange into diastere-omeric spiroiminodihydantoin nucleosides. Nevertheless, the main product of single oxygen oxidation has been found to be 8-oxodGuo and the spiroimin-odihydantoin nucleosides and oxazolone are the secondary oxidation prod-ucts. Its formation occurs via the rearrangement of 4,endoperoxide into 8-hydroperoxy-2’-deoxyguanosine, and subsequent reduction. 8-oxodGuo pos-sess a low oxidation potential and optimal susceptibility to type I and type II oxidation reactions (Figure 3.7).
Furthermore, the presence of 8-oxoGua is an indicator of the presence of oxidative stress within a biological species, as it can be generated by several oxidating agents.
Photophysics fundamentals of
nucleic acids
The different chain reactions and products resulting from the damage due to UV light are rooted in the chemical and structural differences between DNA and RNA. Presumably, due to the fact that at their primordial origin the light conditions were of intense UV radiation [52], nucleic acids (NAs) developed a natural photostability, rendering these molecules the building blocks of Nature [53].
Molecular photostability is a consequence of intermolecular and intramolec-ular factors and even though two molecules absorb light equally, the light-induced inflicted damage might be different. The electronic excitation can initiate photochemical reactions or can be dissipated through radiative pro-cesses (fluorescence or phosphorescence), otherwise internal conversion can convert the excited state to the ground state. If photochemical reactions do not occur because internal conversion is too fast, the molecule will have a short excited state lifetime, thus be photoresistant to UV photodamage. Rapid internal conversion dissipates the electronic energy by converting it to internal energy in the ground state hindering other pathways. Purines and pyrimidines exhibit super short excited state lifetimes, on the other hand, nucleobases derivatives have orders of magnitude longer lifetimes. The key
point governing ultrafast internal conversion is the occurrence of conical in-tersections (CI) connecting the excited state potential energy surface (PES) to the ground state energy surface. The different lifetimes among nucleobases are due to variations in the n,π∗ excited state potential surfaces [54]. Conical
intersections are the crossings of multidimensional potential energy surfaces. Thus, they can only occur where there are regions of the potential energy sur-faces representing a deformation of the molecular structure from the ground state equilibrium geometry [55]. Other compelling properties of NAs are thermodynamic stability, self assembling capability and peculiar electronic structure. DNA even results to be highly impermeable to oxygen [56].
In order to understand the photophysics of RNA and DNA, the excited states dynamics of individual bases need to be taken into account and, sub-sequently, the dynamics of assembled polynucleotides. Each base presents a number of tautomers that complicates the interpretation of photophysical experiments, as the electronic structure of individual tautomers can differ dramatically. Nonetheless, most tautomers are at higher energies than the minimum energy tautomer, thus, they do not occur significantly [17].
An additional level of complexity is the difference in the photophysics of single nucleobases, paired bases, oligonucleotides, polynucleotides and single and double strands. In single nucleobases, a multiexponential behaviour in decay can be observed, and the ground state (GS) is restored in picosec-onds. In the case of single and double strands the photodynamics are more complicated and the excited state population decays with different time com-ponents. A further complication is the dependence of the excited states dy-namics on the excitation wavelength, both for isolated bases and for single-and double-strsingle-anded nucleic acid [53].
Spectroscopic properties As mentioned before, one distinctive feature of each nucleobases is the very low fluorescence quantum yield produced by UV excitation, which vary between Φf= 3×10−5 for uracile and Φf= 2.6×10−4
processes depleting the1ππ∗ state within 200 fs to 1 ps [16]. The fluorescence
lifetime, τf is directly correlated to its fluorescence quantum yield, φf and
to the radiative lifetime τ0 and it cannot be measured directly but it can be
calculated through absorption and emission spectra. The radiative lifetime depends inversely on the oscillator strength of the lowest energy absorption band. Therefore, low fluorescence quantum yields correspond to short excited state lifetimes, hence indicating that the majority of their excited states decays with non radiative pathways [17, 58].
4.1
Isolated bases
Isolated nucleobases are very photostable compounds as they return to the GS in an ultrafast time scale, thus reducing the probability of producing photoproducts from reactive excited states. Internal conversion mechanisms allow ultrafast deactivation, where the excited species transfers its energy to the nuclear vibrational degrees of freedom, deactivating the molecule without emission of photons. This energy transfer process occurs more efficiently near conical intersections, which are nuclear geometries with state degeneracies [59]. The universal mechanism valid for all DNA bases involves a two-step photodeactivation where the population initially goes from an excited bright state ππ∗ through an nπ∗ dark state to the ground state via two conical
intersections [60].
The aim of this section is to provide a summary of the photoproperties of the isolated bases, starting from the lowest energy excited states and moving to their PES, the relevant electronic states, conical intersections, station-ary points and crossing regions. Understanding isolated bases is crucial to interpret the overall functioning of nucleic acids complex mechanisms.
4.1.1
Thymine and Uracil
Thymine and Uracil exhibit very similar behaviour and their differences are mainly faster time constant for Ura and are rooted in a dynamic origin.
The excited states dynamics of these nucleobases are mostly played by the following states: Snπ∗, Sππ∗ and the GS. The lowest energy excited
state is an nπ∗ transition (Snπ∗), whereas Sππ∗ arises from an intense ππ∗
HOMO→LUMO transition. The excited state minimum on S1 for Thymine
and Uracil lie in the Snπ∗, whilst the Sππ∗ minimum lies on the S
2 state.
There are also several conical intersections between S0 and S1, one involving
a stretch and a torsion of a C-C bond and others lying at higher energies. The location of triple (S2/S1/S0) degeneracy among the three states has also
been found [53]. The ultrafast decay of singlet excited states is based on the interplay between the Sππ∗ and Snπ∗ states. Considering figure 4.1,
there are two different decay pathways: the first pathway goes through a plateau with low energy gradient, the other one starts with nuclear relaxation on the S2 surface, leading to a minimum S(πoπ∗). Afterwards, there is a
passage through the S2/S1 intersection; another possible pathway from this
intersection is the indirect relaxation to S0. Both the direct and indirect
decay path are able to yield the Snπ∗ state. Although, the radiationless
decay from this state minimum is not favourable because its a gateway for the triplet state. However, it is still an issue to derive the experimental time constants to the competing pathways as the dynamic results depend sensitively to the method. The solvent has also an important role has it is able to modulate the interplay between Sππ∗ and Snπ∗.
Figure 4.1: Potential energy plot for the ground and excited states of Uracil, and Thymine. (1) Direct decay pathway (2) Indirect decay pathway [53].
Triplet states Another important point of the photophysics of Thy and Ura is the formation of triplet states (3ππ∗), as triplet sensitized
irradia-tion may induce dimerizairradia-tion of neighbouring stacked pyrimidine pairs. The probability of intersystem crossing is given by the spin-orbit coupling con-stant (SOC). The most likely pathway for intersystem crossing from singlet to triplet state is from the 1nπ∗ state as the SOC between 1nπ∗ and 3ππ∗
is high (60 cm−1), accordingly to El-Sayed’s rules [61]. The quantum yield
of triplet formation is wavelength dependent and ranges from 0.4% at 280 nm to 3% at 250 nm [16]. Other pathways yielding a triplet state have been observed to be less favourable.
4.1.2
Cytosine and its derivatives
For Cyt and its derivatives, the lowest energy excited state is a ππ∗
exci-tation (Sππ∗) matching the HOMO→LUMO transition. This state is
desta-bilized in water because of the decrease in dipole moment induced by the S0 →Sππ∗ transition. There are also two excited Snπ∗ states and the
poten-tial energy surface of the excited state is marked by several minima.
The radiationless decay pathways of Cyt can be explained via two conical intersections (named "sofa" and "twist"), and in order to access them the
same energy barrier needs to be overcome [62]. The description of the coni-cal intersections involved in radiationless decay encountered methodologiconi-cal issues and so far the best mechanism, firstly described by Sobolewski et al. involves an ethylenic type S1/S0 conical intersection, involving the torsion of
the ring substituents along a C-C bond (figure 4.2). It is worth to mention that together with radiationless decay, the excited state tautomerization of the amino-oxo tautomer can also take place yielding the imini-oxo tautomer. This product might also form during the decay through an Eth-CI or out of plane CI.
Figure 4.2: Decay pathways for the excited state of Cytosine through the conical intersections [53].
Triplet states The role of triplet state is very relevant as the formation of the cyclobutane pyrimidine dimer has a primary role in the photodamage of nucleic acids. The efficient intersystem crossing rates are backed up by large SOC values and the mixed ππ∗/n
oπ∗ character of the crossing states.
4.1.3
Adenine and its derivatives
The three lowest excited states of Adenine are close in energy and their order is still a matter of debate. They are: a weakly absorbing Snπ∗ state
(HOMO-1→LUMO) and two ππ∗ states, named L
a and Lb. The latter two
excited states correspond, respectively, to a HOMO→LUMO very intense transition and to a combination of HOMO→LUMO+1. The states Snπ∗ and
La are the key of Ade photophysical behaviour and their coupling yields a
single adiabatic minimum on the S1 PES.
The non radiative decays are represented by two CIs: 2E-CI (E:envelope
and 2:atom distorted out of plane) and 6E-CI. The former is more effective
and decay to the ground state is often observed. The role of 6E-CI and
the Snπ∗ and L
b states are still unclear. In figure 4.3 a summary of the
photoexcited decays is shown.
Figure 4.3: Photoexcited decays of Adenine in water solution [53].
4.1.4
Guanine and its derivatives
There are several tautomers of guanine, the most stable isomer is 7H-Gue (1H-7H-2-amino-6-oxopurine) and 9H-Gua (1H-9H-2-amino-6-oxopurine). The three lowest excited electronic state are, similarly to Ade, Snπ∗, L
a and Lb.
State Lais more stable than Lb and it is marked with a HOMO→LUMO
tran-sition; Lb is a combination of transitions (HOMO→LUMO+1 and
HOMO-1→LUMO). The state Lamainly drives the excited state decay and the most
relevant radiationless decay pathway implicate a crossing seam (2E-CI). An
additional conical intersection 6E-CI can also be involved. In figure 4.4, a
Figure 4.4: Scheme of photoexcited decay of Guanine [53].
4.2
Nucleic acid photophysics
The different photophysical behaviour between nucleic acids and its sep-arate components complicates the modelling and understanding of these macromolecules. In fact, DNA needs to be considered a multichromophoric system because of the electronic interactions between nucleobases. These electronic interactions between adjacent bases keep the strands together (Fig-ure 4.5) and can modify the temporal evolution of the excited electronic states, thus further complicating the photophysical analysis of single- and double-stranded DNA. The excited state dynamics depends strictly on molec-ular structure, therefore, nucleobases excited state lifetimes can vary widely from femtoseconds to (sub)picoseconds. Additionally, the absorption bands of the four bases overlap, therefore it is not possible to achieve selective excitation of one specific base.
Figure 4.5: Possible assemblies of nucleobases [58].
In figure 4.6 the differences in deactivation processes between isolated and stacked bases are shown. In single unstacked bases, internal conversion to the electronic ground state, causing the generation of vibrationally hot molecules, is the main excited state depletion. The vibrational energy is dissipated by heat transfer to the surrounding medium on a 1-10 ps timescale.
Stacking interactions reorganize the structures and yield the delocaliza-tion of the excitadelocaliza-tion over several bases or base pairs. The nonradiative processes (internal conversion (IC); intersystem crossing (ISC), and charge transfer (CT)) are followed by vibrational cooling. Hence, UV light absorp-tion is followed by two main relaxaabsorp-tion pathways for excess electronic energy; the excited states either yield photoproducts or decay harmlessly to the elec-tronic ground state. Upon excitation long DNA model systems have shown the formation of long lived excited states localized on a stack of only two bases. Stack bases decay to exciplex states whereas unstacked bases decay via ultrafast internal conversion. These exciplex states are formed by in-terbase charge transfer. Hence, even though the role of base pairing is still unclear, the yields of exciplex formation in base-stacked nucleotides are high (see Table 4.1), and this implies that the decay to exciplex states is the dom-inant decay channel in nucleic acids. The relaxation pathways in DNA are thus determined mainly by the π stacking rather than by the photophysical properties of its building blocks [58, 63].
Dinucloside Yield
ApA 29±5
ApC 27±8
ApG 33±6
ApU 23±9
Table 4.1: Exciplex yields for various diribonucloside monophosphates ApA, ApG, ApU, CpG. Data taken from ref. [63].
Figure 4.6: Summary of photophysical processes after the UV excitation of, respec-tively, isolated unstacked DNA bases and stacked DNA bases. [16].
Excited states dynamics in single stranded DNA
Adjacent bases in DNA are able to shape the stacked geometry of the macromolecule through interactions of their electronic systems. These elec-tronic interactions between strands can modify to a great extent the temporal evolution of the excited electronic states. Single-stranded DNA presents π stacking between neighbouring bases leading to structured domains,
simi-larly to the double helix DNA structure. The study of single-stranded DNA is more selective and easier than double-stranded DNA.
Crespo et al. showed through time-resolved absorption spectra that the amplitudes of long living components depend on the probability of base stacking [64]. These experiments further proved the charge transfer char-acter, thus the formed excimers/exciplexes will exhibit low radiative rate constants and low contributions to fluorescence emission and will disappear on a subnanosecond timescale by charge recombination. Additionally, the charge transfer depends on the type of base. Furthermore, by selectively exciting one specific base in the unstructured section, the 1ππ∗ state rapidly
decays, whereas in a stacked section excitation is delocalized. Bouvier et al. calculated the delocalization of the exciton eigenstates and resulted to be over the entire length of a double helix containing 20 base pairs [65]. How-ever, with the inclusion of conformational (off-diagonal) disorder and the addition of homogenous broadening (diagonal disorder), the excitons results to be delocalized over no more than two bases.
The role of the solvent In aqueous solution, single-stranded sequences are able to form helices, which have a similar conformation to the double helix strands and they are also an ideal model to study the base-stacking effects on excited states dynamics in absence of base pairing. Stranded polynucleotides yield excited states that decay slower than their monomeric equivalents or single base pairs. The role of solvent is complex as, not only it can im-pact excited states dynamic by hydrogen bonding interactions and structural changes, but also might quench internal excitation.
Excited states dynamics in double stranded DNA
The double-helical structure of DNA induce a base stacking in both strands involving a parallel arrangement of heterocycles of neighbouring bases. The double-helical organization is one of the most relevant feature that make DNA one of the most important biomolecules. The stability of
the double helix is assured by hydrogen bonds between bases and electronic interstrand interactions (Figure 4.5). Charge transfer along the strand is also facilitated by the close distance between the bases. These pairing interactions add further complexity to DNA photophysics.
The coupling of1ππ∗ excited state of proximal nucleobases forms Frenkel
exciton states. A Frenkel exciton is defined as “the excited state of a mul-tichromophoric system produced by dipolar coupling of the neutral excited states of individual molecules” [58] and basically they are quantum states arising as linear combination of single-base wavefunctions. The size of the excitation is still unclear but from theoretical calculation it seems that exci-tons are localized on a single base or no more than two bases [58, 63]. The excited state lifetime in DNA is a clear indicator that the rate of nonradiative decay exceeds the rate of radiative decay. Excitons in DNA decay rapidly to exciplexes, which further decay by charge recombination. The Frenkel exciton states rapidly decay to charge transfer states formed between two π-stacked bases. Due to their CT character, these excimer/exciplex states are dark and give little contribution to the total fluorescence. The effect of base stacking influences both the nature of the initial excitation and the following decay pathways.
In conclusion, excited stacked bases initially form excimer/exciplex states with CT character. The decay of these states by charge combination might have a primary role in the photostability of DNA. Although the DNA exciplex lifetimes depend sensitively on base sequence, they proved to be insensitive to base pairing and helix conformation [58, 63].
Several models and experiments have measured experimental parameters and decay pathways [16, 66, 67]. However, additional theoretical calculation and experiments are needed in order to unravel the molecular structure of the long-living states and decay mechanisms, thus leading to a deeper under-standing of the excited states. Another obstacle encountered when studying natural DNA is the fact that, even when extensively purified, they are con-taminated by fluorescent proteins [17].
Experiments and applications
5.1
Advantages and disadvantages
All the techniques and tools elucidated in the previous sections show that UV disinfection has great potential as it is an extremely selective tech-nique; some studies shows that photodynamic treatment of blood samples or blood products constitute a valid procedure to selectively destroy viral contaminants whereas other blood components are spared [68]. Additionally, it seems to be unlikely that pathogens might develop resistance against the cytotoxic action of UV light.
Nonetheless, the use of light has some experimental drawbacks; any light source of any wavelength cannot reach the part of a sample surface in shadow, thus, it will not be completely disinfected as viruses are inactivated only when they are under direct exposure. This can be an issue especially for disinfection of hospital rooms where multiple surfaces might not be reached by UV light. Recently, Lindblad et al. proved this point in a study in which a standard radiometer, emitting UVC light, was employed to disinfect ICU rooms, operated as instructed by the manufacturer. The results showed that places like the wardrobe or the sink, that were partly or completely in shadow, did not capture enough dose to assure 99.99% disinfection [69]. This means that, in order to assure maximum disinfection efficiency, it is required
