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Abstract The development of a suitable irradiation setup is essential for in vitro experiments in photodynamic therapy (PDT). While various irradiation systems have been developed for PDT, only a few offer practical and high‐quality setups for precise and reproducible results in cell culture experiments. This report introduces a cost‐effective illumination setup designed for in vitro photodynamic treatments. The setup consists of a commercially available light‐emitting diode (LED) lamp, a cooling unit, and a specially designed 3D‐printed enclosure to accommodate a multiwell plate insert. The LED lamp is versatile, supporting various irradiation wavelengths and adjustable illumination fields, ensuring consistent and reliable performance. The study evaluates the setup through various parameters, including photon flux density, illumination uniformity, photon distribution across the multiwell plate, and temperature changes during irradiation. In addition, the effectiveness of the LED‐based illumination system is tested by treating mouse mammary breast carcinoma cells (4T1) with Rose Bengal and LED irradiation at around 525 nm. The resulting IC₅₀of 5.2 ± 0.9 μM and a minimum media temperature change of ca. 1.2°C indicate a highly promising LED‐based setup that offers a cost‐effective and technically feasible solution for achieving consistent, reproducible, and uniform irradiation, enhancing research capabilities and potential applications. 

Abstract The (6–4) photolesion is a key photodamage that occurs when two adjacent pyrimidine bases in a DNA strand bond together. To better understand how the absorption of UVB and UVA radiation by the 2‐pyrimidinone moiety in a (6–4) lesion can damage DNA, it is important to study the electronic deactivation mechanism of its 2‐pyrimidinone chromophore. This study employs theoretical (MS‐CASPT2/cc‐pVDZ level) and experimental (steady state and femtosecond broadband spectroscopic) methods to elucidate the photochemical relaxation mechanisms of 2‐(1H)‐pyrimidinone and 1‐methyl‐2‐(1H)‐pyrimidinone in aqueous solution (pH 7.4). In short, excitation at 320 nm leads to the population of the S₁¹(ππ*) state with excess vibrational energy, which relaxes to the S₁¹(ππ*) minimum in one picosecond or less. A trifurcation event in the S₁¹(ππ*) minimum ensued, leading to radiative and nonradiative decay of the population to the ground state or the population of the long‐lived and reactive T₁³(ππ*) state in hundreds of picoseconds. Collectively, the theoretical and experimental results support the idea that in DNA and RNA, the T₁³(ππ*) state of the 2‐pyrimidinone moiety in the (6–4) lesion can further participate in photosensitized chemical reactions increasing DNA and RNA damage. 

Abstract Prolonged ultraviolet exposure results in the formation of cyclobutane pyrimidine dimers (CPDs) in RNA. Consequently, prebiotic photolesion repair mechanisms should have played an important role in the maintenance of the structural integrity of primitive nucleic acids. 2,6‐Diaminopurine is a prebiotic nucleobase that repairs CPDs with high efficiency when incorporated into polymers. We investigate the electronic deactivation pathways of 2,6‐diaminopurine‐2′‐deoxyribose and 9‐methyl‐2,6‐diaminopurine in acetonitrile and aqueous solution to shed light on the photophysical and excited state properties of the 2,6‐diaminopurine chromophore. Evidence is presented that both are photostable compounds exhibiting similar deactivation mechanisms upon the population of the S₁(ππ* L_a) state at 290 nm. The mechanism involves deactivation through the C2‐ and C6‐reaction coordinates and >99% of the excited state population decays through nonradiative pathways involving two conical intersections with the ground state. The radiative and nonradiative lifetimes are longer in aqueous solution compared to acetonitrile. Whileτ₁is similar in both derivatives,τ₂is ca. 1.5‐fold longer in 2,6‐diaminopurine‐2′‐deoxyribose due to a more efficient trapping in the S₁(ππ* L_a) minimum. Therefore, 2,6‐diaminopurine could have accumulated in significant quantities during prebiotic times to be incorporated into non‐canonical RNA and play a significant role in its photoprotection. 

Synthetic biology aims to expand the genetic code by increasing cellular information storage and retrieval. A recent advance is the dTAT1-dNaM unnatural base pair, which is more photo- and thermostable than dTPT3-dNaM while maintaining high efficiency and fidelity in vitro and in vivo. However, the photophysics and cytotoxicity behavior of dTAT1 under UV light have not been investigated. We demonstrate that dTAT1 populates the triplet state upon 390 nm excitation but exhibits minimal cytotoxicity in cells. Analysis of reactive oxygen species indicates that dTAT1 produces a low singlet oxygen quantum yield of 17% while it generates superoxide, a less harmful reactive oxygen species. Its triplet lifetime is 2.7 times shorter than that of dTPT3, contributing to its lower photocytotoxicity. These findings highlight the potential of dTAT1 for safe genetic code expansion and therapeutic applications, providing valuable insights for designing next-generation unnatural nucleosides with minimal impact on cellular health. 

This review provides focused coverage of the photophysical properties of noncanonical and synthetic nucleobases reported over the past decade. It emphasizes key research findings and physical insights gathered for prebiotic and fluorescent nucleobase analogs, sulfur- and selenium-substituted nucleobases, aza-substituted nucleobases, epigenetic nucleobases and their oxidation products, and nucleobases utilized for expanding DNA/RNA to reveal central structure–photophysical property relationships. Further research and development in this emerging field, coupled with machine learning methods, will enable the effective harnessing of nucleobases’ modifications for applications in biotechnology, biomedicine, therapeutics, and even the creation of live semisynthetic organisms. 

Heavy-atom-free photosensitizers (HAF-PSs) have emerged as a new class of photosensitizers aiming to broaden their applicability and versatility across various fields of the photodynamic therapy of cancers. The strategy involves replacing the exocyclic oxygen atoms of the carbonyl groups of established biocompatible organic fluorophores with sulfur, thereby bathochromically shifting their absorption spectra and enhancing their intersystem crossing efficiencies. Despite these advancements, the photophysical attributes and electronic relaxation mechanisms of many of these HAF-PSs remain inadequately elucidated. In this study, we investigate the excited state dynamics and photochemical properties of two promising HAF-PSs, thio-coumarin and thio-acridone. Employing a combination of steady-state and time-resolved techniques from femtoseconds to microseconds, coupled with quantum chemical calculations, we unravel the electronic relaxation mechanisms that give rise to the efficient population of long-lived and reactive triplet states in these HAF-PSs. 

Nucleoside-modified messenger RNA (mRNA) technologies necessarily incorporate N1-methylpseudouridine into the mRNA molecules to prevent the over-stimulation of cytoplasmic RNA sensors. Despite this modification, mRNA concentrations remain mostly determined through the measurement of UV absorbance at 260 nm wavelength (A260). Herein, we report that the N1-methylpseudouridine absorbs approximately 40% less UV light at 260 nm than uridine, and its incorporation into mRNAs leads to the under-estimation of nucleoside-modified mRNA concentrations, with 5%–15% error, in an mRNA-sequence-dependent manner. We therefore examined the RNA quantification methods and developed the mRNACalc webserver. It accounts for the molar absorption coefficient of modified nucleotides at 260 nm wavelength, the RNA composition of the mRNA, and the A260 of the mRNA sample to enable accurate quantification of nucleoside-modified mRNAs. 

It is demonstrated that thieno[3,4-d]pyrimidin-4(3H)-thione is an effective, oxygenation independent, heavy-atom-free photosensitizer against mono-layer of melanoma and cervical cancer cells.