In this study, absorption, fluorescence, synchronous fluorescence, and Raman spectra of nonirradiated and ultraviolet (UV)-irradiated thymine solutions were recorded in order to detect thymine dimer formation. The thymine dimer formation, as a function of irradiation dose, was determined by Raman spectroscopy. In addition, the formation of a mutagenic (6-4) photoproduct was identified by its synchronous fluorescence spectrum. Our spectroscopic data suggest that the rate of conversion of thymine to thymine dimer decreases after 20 min of UV irradiation, owing to the formation of an equilibrium between the thymine dimers and monomers. However, the formation of the (6-4) photoproduct continued to increase with UV irradiation. In addition, the Raman spectra of nonirradiated and irradiated calf thymus DNA were recorded, and the formation of thymine dimers was detected. The spectroscopic data presented make it possible to determine the mechanism of thymine dimer formation, which is known to be responsible for the inhibition of DNA replication that causes bacteria inactivation.In the United States alone, each year 2.8 million antibiotic-resistant infections occur that cause about 35,000 deaths (
1). Such resistant organisms are often acquired in the hospital (
2) and may well be from contaminated surfaces (
3). Ultraviolet (UV) light has become a critical means to control some of these antibiotic-resistant organisms and to prevent them from infecting vulnerable patients (
4). Therefore, improving our understanding of the microbiologic effects of UV light has become more important. Further, UV light also has an antimicrobial effect on the RNA of viruses, including coronaviruses (
5), which further increases our need to understand the chemical basis for the biological effects of UV light. DNA damage, such as deamination, oxidative damage, strand breaks, and dimer formation, can hinder normal functioning of a cell, prevent DNA replication, and cause cell death (
6). UV-induced DNA damage results mainly in damage to pyrimidine bases, thus inducing the formation of cyclobutane pyrimidine dimers (CPDs), pyrimidine(6-4)pyrimidone photoproducts, and Dewar isomers (
7). It is widely believed, but not previously shown spectroscopically, that inactivation of bacteria by UV irradiation is caused by the dissociation of the double bond of the thymine base of the bacterial DNA followed by the formation of a single bond formed between two adjacent thymine bases, which results in the formation of thymine CPDs (
8). These dimers dissociate the hydrogen bonds between bases of complementary DNA strands and consequently inhibit the replication of DNA (
9,
10). It has been reported that the most abundant photoproduct formed after UV irradiation is the thymine dimer T-T, followed by thymine cytosine dimer T-C, thymine cytosine (6-4) photoproduct T(6-4)C, and thymine (6-4) photoproduct T(6-4)T (
11,
12). Many studies have suggested that the thymine (6-4) photoproduct is as mutagenic as the thymine CPD (
13,
14); however, the (6-4) photoproduct is formed with a smaller yield (
12). shows the structure of thymine, thymine CPD, and (6-4) photoproduct (
15). Minor UV photoproducts include cytosine dimers (C-C), cytosine thymine dimers (C-T), and their respective (6-4) adducts, which are formed in much smaller quantities (
12).
Open in a separate window(
A) Structure of thymine, thymine dimer (CPD), and (6-4) photoproduct. (
B) Absorption spectrum of thymine solution.Purine bases are considered to be virtually immune to UV light. The formation of adenine dimers and adenine thymine photoproducts has also been observed; however, their yield is orders of magnitude smaller than that of the pyrimidine dimers and their adducts (
16,
17).Previous studies have proposed that the thymine CPD, in frozen thymine solutions, is formed by the combination of the excited singlet state of the thymine monomers (
18), whereas the (6-4) photoproduct is formed from an oxetane intermediate (
7). As a result, thymine dimers can dissociate and reconvert into thymine monomers by UV irradiation. However, we find, in accordance with previous studies, that the (6-4) photoproducts are not converted to thymine monomers by UV irradiation (
13). When the (6-4) photoproduct is irradiated with 313-nm light it is reversibly converted to another UV photoproduct, known as Dewar isomer, which converts back to the (6-4) photoproduct upon irradiation with 240-nm light (
19).Spectroscopic analysis of bacteria provides a fast and cost-effective method for the determination of bacterial strains and, in addition, allows for the detection of live and dead bacteria after UV irradiation (
20,
21). In our study, aqueous solutions of thymine and DNA were irradiated with UV light and changes in their Raman spectra were compared, before and after irradiation, in order to detect UV-induced photoproducts. Aqueous thymine solutions were frozen at 240 K and then irradiated with 254-nm UV light. The absorption spectrum of thymine is shown in . When aqueous thymine solution is frozen, water molecules start to crystallize into a hexagonal structure in order to attain the lowest energy configuration. The water crystallization process (ice formation) excludes thymine molecules and consequently leads to separation of the solvent and solute components (
22). Therefore, as the water crystallizes, the thymine solution becomes more concentrated and crystallizes into thymine monohydrate crystals (
23). The preferred structural orientation of these crystals is such that the thymine monomers are stacked on top of each other, yielding the perfect structural orientation for dimer formation. This arrangement of the frozen thymine monomers mimics the arrangement of the thymine monomers in DNA strands, which also form thymine dimers upon UV irradiation at room temperature (
24).In agreement with previous studies (
25,
26), our experiments show that when thymine solution is irradiated with UV light the absorption band intensity at 260 nm decreases due to the dissociation of the thymine monomers and the formation of photoproducts. The thymine dimer, in contrast to the thymine monomer, does not absorb at 260 nm, because the 260-nm absorbing C=C is converted to C–C, while the (6-4) photoproduct exhibits an absorption maximum at 315 nm. To confirm the formation of thymine dimers, after UV irradiation at 240 K we reirradiated the irradiated solution at 300 K. We observed () that the absorption band at 260 nm increases with thymine irradiation time at room temperature, owing to the dissociation of thymine dimers and their conversion to thymine monomers.
Open in a separate window(
A) Absorption spectrum of thymine solution irradiated with 254-nm light at 240 K and reirradiated at 300 K for 10, 20, and 30 min. (
B) Change in OD at 263.8 nm, after UV irradiation at 240 K followed by irradiation at 300 K, as a function of irradiation time (minutes).Thymine solution exhibits a weak fluorescence with maximum intensity at 325 nm, when excited at 260 nm (
27), and upon UV irradiation with 254-nm light at 240 K the thymine fluorescence decreases. We also observed that the (6-4) photoproduct fluoresces with a maximum at 375 nm, when excited at 315 nm, whereas no thymine dimer fluorescence was observed. Therefore, to detect thymine dimers which were not previously identified spectroscopically we recorded the Raman spectra of thymine before and after irradiation.Raman spectroscopy is a well-known powerful method for the study of biological molecules, including amino acids, proteins, nucleic acids, lipids, and other molecules (
28–
33). This fast and noninvasive technique, based on the inelastic scattering of monochromatic light from a molecule, provides a structural fingerprint by recording their vibrational and rotational transitions. As a result, it is widely used for structural analysis and the identification of molecules.
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