The kinetic landscape of an RNA-binding protein in cells.

Gene expression in higher eukaryotic cells orchestrates interactions between thousands of RNA-binding proteins (RBPs) and tens of thousands of RNAs1. The kinetics by which RBPs bind to and dissociate from their RNA sites are critical for the coordination of cellular RNA-protein interactions2. However, these kinetic parameters have not been experimentally measured in cells. Here we show that time-resolved RNA-protein cross-linking with a pulsed femtosecond ultraviolet laser, followed by immunoprecipitation and high-throughput sequencing, allows the determination of binding and dissociation kinetics of the RBP DAZL for thousands of individual RNA-binding sites in cells. This kinetic cross-linking and immunoprecipitation (KIN-CLIP) approach reveals that DAZL resides at individual binding sites for time periods of only seconds or shorter, whereas the binding sites remain DAZL-free for markedly longer. The data also indicate that DAZL binds to many RNAs in clusters of multiple proximal sites. The effect of DAZL on mRNA levels and ribosome association correlates with the cumulative probability of DAZL binding in these clusters. Integrating kinetic data with mRNA features quantitatively connects DAZL-RNA binding to DAZL function. Our results show how kinetic parameters for RNA-protein interactions can be measured in cells, and how these data link RBP-RNA binding to the cellular function of RBPs.

Photophysical Characterization of Isoguanine in a Prebiotic-Like Environment.

It is intriguing how a mixture of organic molecules survived the prebiotic UV fluxes and evolved into the actual genetic building blocks. Scientists are trying to shed light on this issue by synthesizing nucleic acid monomers and their analogues under prebiotic Era-like conditions and by exploring their excited state dynamics. To further add to this important body of knowledge, this study discloses new insights into the photophysical properties of protonated isoguanine, an isomorph of guanine, using steady-state and femtosecond broadband transient absorption spectroscopies, and quantum mechanical calculations. Protonated isoguanine decays in ultrafast time scales following 292 nm excitation, consistently with the barrierless paths connecting the bright S1 (ππ*) state with different internal conversion funnels. Complementary calculations for neutral isoguanine predict similar photophysical properties. These results demonstrate that protonated isoguanine can be considered photostable in contrast to protonated guanine, which exhibits 40-fold longer excited state lifetimes.

Increased Photostability of the Integral mRNA Vaccine Component N1 -Methylpseudouridine Compared to Uridine.

N1 -Methylation of pseudouridine (m1 ψ) replaces uridine (Urd) in several therapeutics, including the Moderna and BioNTech-Pfizer COVID-19 vaccines. Importantly, however, it is currently unknown if exposure to electromagnetic radiation can affect the chemical integrity and intrinsic stability of m1 ψ. In this study, the photochemistry of m1 ψ is compared to that of uridine by using photoirradiation at 267 nm, steady-state spectroscopy, and quantum-chemical calculations. Furthermore, femtosecond transient absorption measurements are collected to delineate the electronic relaxation mechanisms for both nucleosides under physiologically relevant conditions. It is shown that m1 ψ exhibits a 12-fold longer 1 ππ* decay lifetime than uridine and a 5-fold higher fluorescence yield. Notably, however, the experimental results also demonstrate that most of the excited state population in both molecules decays back to the ground state in an ultrafast time scale and that m1 ψ is 6.7-fold more photostable than Urd following irradiation at 267 nm.

Excited state dynamics of 2'-deoxyisoguanosine and isoguanosine in aqueous solution.

Photostability is thought to be an inherent property of nucleobases required to survive the extreme ultraviolet radiation conditions of the prebiotic era. Previous studies have shown that absorption of ultraviolet radiation by the canonical nucleosides results in ultrafast internal conversion to the ground state, demonstrating that these nucleosides efficiently dissipate the excess electronic energy to the environment. In recent years, studies on the photophysical and photochemical properties of nucleobase derivatives have revealed that chemical substitution influences the electronic relaxation pathways of purine and pyrimidine nucleobases. It has been suggested that amino or carbonyl substitution at the C6 position could increase the photostability of the purine derivatives more than the substitution at the C2 position. This investigation aims to elucidate the excited state dynamics of 2'-deoxyisoguanosine (dIsoGuo) and isoguanosine (IsoGuo) in aqueous solution at pH 7.4 and 1.4, which contain an amino group at the C6 position and a carbonyl group at the C2 position of the purine chromophore. The study of these derivatives is performed using absorption and emission spectroscopies, broadband transient absorption spectroscopy, and density functional and time-dependent density functional levels of theory. It is shown that the primary relaxation mechanism of dIsoGuo and IsoGuo involves nonradiative decay pathways, where the population decays from the S1(ππ*) state through internal conversion to the ground state via two relaxation pathways with lifetimes of hundreds of femtoseconds and less than 2 ps, making these purine nucleosides photostable in aqueous solution.

Photostability of 2,6-diaminopurine and its 2'-deoxyriboside investigated by femtosecond transient absorption spectroscopy.

Ultraviolet radiation (UVR) from the sun is essential for the prebiotic syntheses of nucleotides, but it can also induce photolesions such as the cyclobutane pyrimidine dimers (CPDs) to RNA or DNA oligonucleotide in prebiotic Earth. 2,6-Diaminopurine (26DAP) has been proposed to repair CPDs in high yield under prebiotic conditions and be a key component in enhancing the photostability of higher-order prebiotic DNA structures. However, its electronic relaxation pathways have not been studied, which is necessary to know whether 26DAP could have survived the intense UV fluxes of the prebiotic Earth. We investigate the electronic relaxation mechanism of both 26DAP and its 2'-deoxyribonucleoside (26DAP-d) in aqueous solution using steady-state and femtosecond transient absorption measurements that are complemented with electronic-structure calculations. The results demonstrate that both purine derivatives are significantly photostable to UVR. It is shown that upon excitation at 287 nm, the lowest energy 1ππ* state is initially populated. The population then branches following two relaxation coordinates in the 1ππ* potential energy surface, which are identified as the C2- and C6-relaxation coordinates. The population following the C6-coordinate internally converts to the ground state nonradiatively through a nearly barrierless conical intersection within 0.7 ps in 26DAP or within 1.1 ps in 26DAP-d. The population that follows the C2-relaxation coordinate decays back to the ground state by a combination of nonradiative internal conversion via a conical intersection and fluorescence emission from the 1ππ* minimum in 43 ps and 1.8 ns for the N9 and N7 tautomers of 26DAP, respectively, or in 70 ps for 26DAP-d. Fluorescence quantum yields of 0.037 and 0.008 are determined for 26DAP and 26DAP-d, respectively. Collectively, it is demonstrated that most of the excited state population in 26DAP and 26DAP-d decays back to the ground state via both nonradiative and radiative relaxation pathways. This result lends support to the idea that 26DAP could have accumulated in large enough quantities during the prebiotic era to participate in the formation of prebiotic RNA or DNA oligomers and act as a key component in the protection of the prebiotic genetic alphabet.

On the Photostability of Cyanuric Acid and Its Candidature as a Prebiotic Nucleobase.

Cyanuric acid is a triazine derivative that has been identified from reactions performed under prebiotic conditions and has been proposed as a prospective precursor of ancestral RNA. For cyanuric acid to have played a key role during the prebiotic era, it would have needed to survive the harsh electromagnetic radiation conditions reaching the Earth's surface during prebiotic times (≥200 nm). Therefore, the photostability of cyanuric acid would have been crucial for its accumulation during the prebiotic era. To evaluate the putative photostability of cyanuric acid in water, in this contribution, we employed density functional theory (DFT) and its time-dependent variant (TD-DFT) including implicit and explicit solvent effects. The calculations predict that cyanuric acid has an absorption maximum at ca. 160 nm (7.73 eV), with the lowest-energy absorption band extending to ca. 200 nm in an aqueous solution and exhibiting negligible absorption at longer wavelengths. Excitation of cyanuric acid at 160 nm or longer wavelengths leads to the population of S5,6 singlet states, which have ππ* character and large oscillator strengths (0.8). The population reaching the S5,6 states is expected to internally convert to the S1,2 states in an ultrafast time scale. The S1,2 states, which have nπ* character, are predicted to access a conical intersection with the ground state in a nearly barrierless fashion (ca. ≤ 0.13 eV), thus efficiently returning the population to the ground state. Furthermore, based on calculated spin-orbit coupling elements of ca. 6 to 8 cm-1, the calculations predict that intersystem crossing to the triplet manifold should play a minor role in the electronic relaxation of cyanuric acid. We have also calculated the vertical ionization energy of cyanuric acid at 8.2 eV, which predicts that direct one-photon ionization of cyanuric acid should occur at ca. 150 nm. Collectively, the quantum-chemical calculations predict that cyanuric acid would have been highly photostable under the solar radiation conditions reaching the Earth's surface during the prebiotic era in an aqueous solution. Of relevance to the chemical origin of life and RNA-first theories, these observations lend support to the idea that cyanuric acid could have accumulated in large quantities during the prebiotic era and thus strengthens its candidature as a relevant prebiotic nucleobase.

Electronic Relaxation Pathways in Heavy-Atom-Free Photosensitizers Absorbing Near-Infrared Radiation and Exhibiting High Yields of Singlet Oxygen Generation.

Heavy-atom-free photosensitizers (HAF-PSs) based on thionation of carbonyl groups of readily accessible organic compounds are rapidly emerging as a versatile class of molecules. However, their photochemical properties and electronic relaxation mechanisms are currently unknown. Investigating the excited-state dynamics is essential to understand their benefits and limitations and to develop photosensitizers with improved photochemical properties. Herein, the photochemical and electronic-structure properties of two of the most promising HAF-PSs developed to date are revealed. It is shown that excitation of thio-4-(dimethylamino)naphthalamide and thionated Nile Red with near-infrared radiation leads to the efficient population of the triplet manifold through multiple relaxation pathways in hundreds of femtoseconds. The strong singlet-triplet couplings in this family of photosensitizers should enable a broad range of applications, including in photodynamic therapy, photocatalysis, photovoltaics, organic LEDs, and photon up-conversion.

Femtosecond intersystem crossing to the reactive triplet state of the 2,6-dithiopurine skin cancer photosensitizer.

Site-selected sulfur-substituted nucleobases are a class of all organic, heavy-atom-free photosensitizers for photodynamic therapy applications that exhibit excellent photophysical properties such as strong absorption in the ultraviolet-A region of the electromagnetic spectrum, near-unity triplet yields, and a high yield of singlet oxygen generation. Recent investigations on doubly thionated nucleobases, 2,4-dithiothymine, 2,4-dithiouracil, and 2,6-dithiopurine, demonstrated that these set of dithionated nucleobases outperform the photodynamic efficacy exhibit by 4-thiothymidine-the most widely studied singly substituted thiobase to date. Out of the three dithionated nucleobases, 2,6-dithiopurine was shown to be the most effective, exhibiting inhibition of cell proliferation of up to 63% when combined with a low UVA dose of 5 J cm-2. In this study, we elucidated the electronic relaxation pathways leading to the population of the reactive triplet state of 2,6-dithiopurine. 2,6-Dithiopurine populates the triplet manifold in less than 150 fs, reaching the nπ* triplet state minimum within a lifetime of 280 ± 50 fs. Subsequently, the population in the nπ* triplet state minimum internally converts to the long-lived ππ* triplet state within a lifetime of 3 ± 1 ps. The relatively slow internal conversion lifetime is associated with major conformational relaxation in going from the nπ* to ππ* triplet state minimum. A unity triplet yield of 1.0 ± 0.1 is measured.

Excited state dynamics of 7-deazaguanosine and guanosine 5'-monophosphate.

Minor structural modifications to the DNA and RNA nucleobases have a significant effect on their excited state dynamics and electronic relaxation pathways. In this study, the excited state dynamics of 7-deazaguanosine and guanosine 5'-monophosphate are investigated in aqueous solution and in a mixture of methanol and water using femtosecond broadband transient absorption spectroscopy following excitation at 267 nm. The transient spectra are collected using photon densities that ensure no parasitic multiphoton-induced signal from solvated electrons. The data can be fit satisfactorily using a two- or three-component kinetic model. By analyzing the results from steady-state, time-resolved, computational calculations, and the methanol-water mixture, the following general relaxation mechanism is proposed for both molecules, Lb → La → 1πσ*(ICT) → S0, where the 1πσ*(ICT) stands for an intramolecular charge transfer excited singlet state with significant πσ* character. In general, longer lifetimes for internal conversion are obtained for 7-deazaguanosine compared to guanosine 5'-monophosphate. Internal conversion of the 1πσ*(ICT) state to the ground state occurs on a similar time scale of a few picoseconds in both molecules. Collectively, the results demonstrate that substitution of a single nitrogen atom for a methine (C-H) group at position seven of the guanine moiety stabilizes the 1ππ* Lb and La states and alters the topology of their potential energy surfaces in such a way that the relaxation dynamics in 7-deazaguanosine are slowed down compared to those in guanosine 5'-monophosphate but not for the internal conversion of 1πσ*(ICT) state to the ground state.

Excited State Lifetimes of Sulfur-Substituted DNA and RNA Monomers Probed Using the Femtosecond Fluorescence Up-Conversion Technique.

Sulfur-substituted DNA and RNA nucleobase derivatives (a.k.a., thiobases) are an important family of biomolecules. They are used as prodrugs and as chemotherapeutic agents in medical settings, and as photocrosslinker molecules in structural-biology applications. Remarkably, excitation of thiobases with ultraviolet to near-visible light results in the population of long-lived and reactive triplet states on a time scale of hundreds of femtoseconds and with near-unity yields. This efficient nonradiative decay pathway explains the vanishingly small fluorescence yields reported for the thiobases and the scarcity of fluorescence lifetimes in the literature. In this study, we report fluorescence lifetimes for twelve thiobase derivatives, both in aqueous solution at physiological pH and in acetonitrile. Excitation is performed at 267 and 362 nm, while fluorescence emission is detected at 380, 425, 450, 525, or 532 nm. All the investigated thiobases reveal fluorescence lifetimes that decay in a few hundreds of femtoseconds and with magnitudes that depend and are sensitive to the position and degree of sulfur-atom substitution and on the solvent environment. Interestingly, however, three thiopyrimidine derivatives (i.e., 2-thiocytidine, 2-thiouridine, and 4-thiothymidine) also exhibit a small amplitude fluorescence component of a few picoseconds in aqueous solution. Furthermore, the N-glycosylation of thiobases to form DNA or RNA nucleoside analogues is demonstrated as affecting their fluorescence lifetimes. In aqueous solution, the fluorescence decay signals exciting at 267 nm are equal or slower than those collected exciting at 362 nm. In acetonitrile, however, the fluorescence decay signals recorded upon 267 nm excitation are, in all cases, faster than those measured exciting at 362 nm. A comparison to the literature values show that, while both the DNA and RNA nucleobase and thiobase derivatives exhibit sub-picosecond fluorescence lifetimes, the 1ππ* excited-state population in the nucleobase monomers primarily decay back to the ground state, whereas it predominantly populates long-lived and reactive triplet states in thiobase monomers.

Tracking the origin of photostability in purine nucleobases: the photophysics of 2-oxopurine.

This work scrutinizes the relaxation mechanism of 2-oxopurine. Contrary to its ancestor, purine, which is a UVC chromophore, 2-oxopurine shows a red-shifted absorption spectrum centered in the UVA region. In 2-oxopurine, relaxation along the ππ* spectroscopic state directs the population from the Franck-Condon (FC) region towards a minimum, which acts as a crossroad for the further decay of the system either to triplet states or, alternatively, to the ground state through a C6-puckered S1/S0 funnel. A comparison of the optical properties and excited state potential energy surfaces of purine, 2-oxopurine, 2-aminopurine, 6-oxopurine and adenine, allows establishing how the position and nature of substituent tune the photophysics of purine. For this series, we conclude that both C2 and C6 substitution redshift the absorption spectrum of purine, with 2-oxo substitution exhibiting the largest shift. An important exception is the canonical nucleobase adenine, which presents a blue shifted absorption spectrum. The topography of purine's ππ* potential energy surface experiences major changes when functionalized at the C6 position. In particular, the disappearance of the minimum along the ππ* potential energy surface efficiently funnels the excited state population from the FC region to the ground state and increases the photostability of 6-aminopurine (adenine) and 6-oxopurine (hypoxanthine) nucleobases.

Heavy-Atom-Substituted Nucleobases in Photodynamic Applications: Substitution of Sulfur with Selenium in 6-Thioguanine Induces a Remarkable Increase in the Rate of Triplet Decay in 6-Selenoguanine.

Sulfur substitution of carbonyl oxygen atoms of DNA/RNA nucleobases promotes ultrafast intersystem crossing and near-unity triplet yields that are being used for photodynamic therapy and structural-biology applications. Replacement of sulfur with selenium or tellurium should significantly red-shift the absorption spectra of the nucleobases without sacrificing the high triplet yields. Consequently, selenium/tellurium-substituted nucleobases are thought to facilitate treatment of deeper tissue carcinomas relative to the sulfur-substituted analogues, but their photodynamics are yet unexplored. In this contribution, the photochemical relaxation mechanism of 6-selenoguanine is elucidated and compared to that of the 6-thioguanine prodrug. Selenium substitution leads to a remarkable enhancement of the intersystem crossing lifetime both to and from the triplet manifold, resulting in an efficiently populated, yet short-lived triplet state. Surprisingly, the rate of triplet decay in 6-selenoguanine increases by 835-fold compared to that in 6-thioguanine. This appears to be an extreme manifestation of the classical heavy-atom effect in organic photochemistry, which challenges conventional wisdom.

Photochemical relaxation pathways of S6-methylthioinosine and O6-methylguanosine in solution.

S6-Methylthioinosine and O6-methylguanosine are byproducts resulting from the enzymatic reactions of sulfur-substituted prodrugs in cells and from the interaction of alkylating agents with cellular DNA, respectively. Their photochemistry has not been investigated, and it is currently unknown whether light absorption by these byproducts may pose any threat to the cell. In this contribution, their photoinduced processes upon absorption of UVB radiation are reported using broadband transient absorption spectroscopy. Plausible electronic relaxation mechanisms are proposed for both biological molecules, which are supported by steady-state absorption and emission measurements, and by singlet and triplet vertical excitation energies performed on a large subset of ground-state optimized conformational isomers in solution. The results are compared to the body of knowledge gathered in the scientific literature about the light-induced processes in the sulfur-substituted and canonical purine monomers. In particular, it is shown that S6-methylation decreases the rate to populate the lowest-energy triplet state and blueshifts the ground-state absorption spectrum compared to those for the sulfur-substituted prodrugs and for the 6-thioguanosine metabolite. Similarly, O6-methylation decreases the rate of internal conversion to the ground state observed in the guanine monomers by more than 10-fold in acetonitrile and 40-fold in aqueous solution, while it redshifts the ground-state absorption spectrum. Collectively, this investigation provides relevant new insights about the relationship between structural modifications of the purine chromophore and the electronic relaxation mechanisms in this important group of biological molecules.

Decoding the Molecular Basis for the Population Mechanism of the Triplet Phototoxic Precursors in UVA Light-Activated Pyrimidine Anticancer Drugs.

The photosensitization of DNA by thionucleosides is a promising photo-chemotherapeutic treatment option for a variety of malignancies. DNA metabolization of thionated prodrugs can lead to cell death upon exposure to a low dose of UVA light. The exact mechanisms of thionucleoside phototoxicity are still not fully understood. In this work, we have combined femtosecond broadband transient absorption experiments with state-of-the-art molecular simulations to provide mechanistic insights into the ultrafast and efficient population of the triplet state in the UVA-activated pyrimidine anticancer drug 4-thiothymine. The triplet state is thought to act as a precursor to DNA lesions and the reactive oxygen species responsible for 4-thiothymine photocytotoxicity. The electronic-structure and mechanistic results presented in this contribution reveal key molecular design criteria that can assist in developing alternative chemotherapeutic agents that may overcome some of the primary deficiencies of classical photosensitizers.

2-Thiouracil intersystem crossing photodynamics studied by wavelength-dependent photoelectron and transient absorption spectroscopies.

Single-atom substitution within a natural nucleobase-such as replacing oxygen by sulfur in uracil-can result in drastic changes in the relaxation dynamics after UV excitation. While the photodynamics of natural nucleobases like uracil are dominated by pathways along singlet excited states, the photodynamics of thiobases like 2-thiouracil populate the triplet manifold with near unity quantum yield. In the present study, a synergistic approach based on time-resolved photoelectron spectroscopy (TRPES), time-resolved absorption spectroscopy (TRAS), and ab initio computations has been particularly successful at unraveling the underlying photophysical principles and describing the dissimilarities between the natural and substituted nucleobases. Specifically, we find that varying the excitation wavelength leads to differences between gas-phase and condensed-phase experimental results. Systematic trends are observed in the intersystem crossing time constants with varying excitation wavelength, which can be readily interpreted in the context of ab initio calculations performed both in vacuum and including solvent effects. Thus, the combination of TRPES and TRAS experiments with high-level computational techniques allows us to characterize the topology of the potential energy surfaces defining the relaxation dynamics of 2-thiouracil in both gas and condensed phases, as well as investigate the accessibility of conical intersections and crossings, and potential energy barriers along the associated relaxation coordinates.

Photochemical Relaxation Pathways in Dinitropyrene Isomer Pollutants.

Dinitropyrenes are polycyclic aromatic pollutants prevalent in the environment. While their transformations by sunlight in the environment have been documented, the effect that the nitro-group substitution pattern has on the relaxation pathways has not been extensively studied. In this contribution, the steady-state and femtosecond-to-microsecond excited-state dynamics of 1,3-dinitropyrene and 1,8-dinitropyrene isomers are investigated upon visible light excitation at 425 nm and compared with those recently reported for the 1,6-dinitropyrene isomer. The experimental results are complemented with ground- and excited-state density functional calculations. It is shown that excitation at 425 nm results in the ultrafast branching of the excited-state population in the S1 state to populate the triplet state in ca. 90% yield and to form a nitropyrenoxy radical in less than 10% yield. In addition, the position of the NO2 group does not affect significantly the excited-state relaxation mechanism, while it does influence the absorption and fluorescence spectra, the fluorescence, triplet, singlet oxygen, and photodegradation yields, as well as the relative yield of radical formation. Radical formation is implicated in the photodegradation of these pollutants, while in the presence of hydrogen donors, direct reactions from the triplet state are also observed.

Photorelaxation and Photorepair Processes in Nucleic and Amino Acid Derivatives.

Understanding the fundamental interaction between electromagnetic radiation and matter is essential for a large number of phenomena, with significance to civilization.[...].

Excited-State Dynamics of the Thiopurine Prodrug 6-Thioguanine: Can N9-Glycosylation Affect Its Phototoxic Activity?

6-Thioguanine, an immunosuppressant and anticancer prodrug, has been shown to induce DNA damage and cell death following exposure to UVA radiation. Its metabolite, 6-thioguanosine, plays a major role in the prodrug's overall photoreactivity. However, 6-thioguanine itself has proven to be cytotoxic following UVA irradiation, warranting further investigation into its excited-state dynamics. In this contribution, the excited-state dynamics and photochemical properties of 6-thioguanine are studied in aqueous solution following UVA excitation at 345 nm in order to provide mechanistic insight regarding its photochemical reactivity and to scrutinize whether N9-glycosylation modulates its phototoxicity in solution. The experimental results are complemented with time-dependent density functional calculations that include solvent dielectric effects by means of a reaction-field solvation model. UVA excitation results in the initial population of the S2(ππ*) state, which is followed by ultrafast internal conversion to the S1(nπ*) state and then intersystem crossing to the triplet manifold within 560 ± 60 fs. A small fraction (ca. 25%) of the population that reaches the S1(nπ*) state repopulates the ground state. The T1(ππ*) state decays to the ground state in 1.4 ± 0.2 µs under N2-purged conditions, using a 0.2 mM concentration of 6-thioguanine, or it can sensitize singlet oxygen in 0.21 ± 0.02 and 0.23 ± 0.02 yields in air- and O2-saturated solution, respectively. This demonstrates the efficacy of 6-thioguanine to act as a Type II photosensitizer. N9-glycosylation increases the rate of intersystem crossing from the singlet to triplet manifold, as well as from the T1(ππ*) state to the ground state, which lead to a ca. 40% decrease in the singlet oxygen yield under air-saturated conditions. Enhanced vibronic coupling between the singlet and triplet manifolds due to a higher density of vibrational states is proposed to be responsible for the observed increase in the rates of intersystem crossing in 6-thioguanine upon N9-glycosylation.

Unintended Consequences of Expanding the Genetic Alphabet.

The base pair d5SICS·dNaM was recently reported to incorporate and replicate in the DNA of a modified strain of Escherichia coli, thus making the world's first stable semisynthetic organism. This newly expanded genetic alphabet may allow organisms to store considerably more information in order to translate proteins with unprecedented enzymatic activities. Importantly, however, there is currently no knowledge of the photochemical properties of d5SICS or dNaM-properties that are central to the chemical integrity of cellular DNA. In this contribution, it is shown that excitation of d5SICS or dNaM with near-visible light leads to efficient trapping of population in the nucleoside's excited triplet state in high yield. Photoactivation of these long-lived, reactive states is shown to photosensitize cells, leading to the generation of reactive oxygen species and to a marked decrease in cell proliferation, thus warning scientists of the potential phototoxic side effects of expanding the genetic alphabet.

Can a Six-Letter Alphabet Increase the Likelihood of Photochemical Assault to the Genetic Code?

In 2014, two unnatural nucleosides, d5SICS and dNaM, were shown to selectively base pair and replicate with high fidelity in a modified strain of E. coli, thus effectively expanding its genetic alphabet from four to six letters. More recently, a significant reduction in cell proliferation was reported in cells cultured with d5SICS, and putatively with dNaM, upon exposure to brief periods of near-visible radiation. The photosensitizing properties of the lowest-energy excited triplet state of both d5SICS and dNaM were implicated in their cytotoxicity. Importantly, however, the excited-state mechanisms by which near-visible excitation populates the triplet states of d5SICS and dNaM are currently unknown. In this study, steady-state and time-resolved spectroscopies are combined with quantum-chemical calculations in order to reveal the excited-state relaxation mechanisms leading to efficient population of the triplet states in these unnatural nucleosides in solution. It is shown that excitation of d5SICS or dNaM with near-visible light leads overwhelmingly to ultrafast population of their triplet states on the femtosecond time scale. The results presented in this work lend strong support to the proposal that photoexcitation of these unnatural nucleosides can accelerate oxidatively generated damage to DNA and other biomolecules within the cellular environment.