The mRNACalc webserver accounts for the N1-methylpseudouridine hypochromicity to enable precise nucleoside-modified mRNA quantification.

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.

Photochemical Stability of 5-Methylcytidine Relative to Cytidine: Photophysical Insight for mRNA Therapeutic Applications.

5-Methylcytidine (5mCyd) has recently been investigated with renewed interest in its utilization in mRNA therapeutics. However, its photostability following exposure to electromagnetic radiation has been overlooked. This Letter compares the photostability and excited-state dynamics of 5mCyd with those of the canonical RNA nucleoside, cytidine (Cyd), using steady-state and femtosecond transient absorption spectroscopy under physiologic conditions. 5mCyd is shown to have a 5-fold higher fluorescence yield and a 5-fold longer 1ππ* excited-state decay lifetime. Importantly, however, the excited-state population in 5mCyd decays primarily by internal conversion, with a photodegradation rate 3 times smaller than that in Cyd. In Cyd, the population of a 1nπ* state with a lifetime of ca. 45 ps is implicated in the formation 6-hydroxycytidine and other photoproducts.

Excited State Dynamics of Dibenzothiophene Derivatives.

Polycyclic aromatic sulfur heterocycles are environmental pollutants formed from incomplete combustion processes and crude oil spills. Their excited state dynamics are not understood. Herein, femtosecond transient absorption is combined with steady-state spectroscopy and computational methods to elucidate the relaxation mechanisms of three dibenzothiophene derivatives. The low-energy singlet and triplet states all have ππ* character in the Franck-Condon region, and two minima were located in the S1 surface. Excitation at 320 nm populates their S1 state directly, which relaxes with lifetimes ranging from 4 to 13 ps. Most of the S1 population undergoes efficient intersystem crossing to the triplet state with lifetimes ranging from 820 ± 50 to 900 ± 30 ps. The compounds exhibit negligible nonradiative internal conversion, low fluorescence yields of 1.2 to 1.6%, and triplet yields of ca. 98%. Linear interpolation of internal coordinates reveals the chemical basis for relaxing the spin-forbidden intersystem crossing in these π-aromatic systems.

Resolving Ultrafast Photoinitiated Dynamics of the Hachimoji 5-aza-7-deazaguanine Nucleobase: Impact of Synthetically Expanding the Genetic Alphabet†.

The guanine derivative, 5-aza-7-deazaguanine (5N7C G) has recently been proposed as one of four unnatural bases, termed Hachimoji (8-letter) to expand the genetic code. We apply steady-state and time-resolved spectroscopy to investigate its electronic relaxation mechanism and probe the effect of atom substitution on the relaxation mechanism in polar protic and polar aprotic solvents. Mapping of the excited state potential energy surfaces is performed, from which the critical points are optimized by using the state-of-art extended multi-state complete active space second-order perturbation theory. It is demonstrated that excitation to the lowest energy 1 ππ* state of 5N7C G results in complex dynamics leading to ca. 10- to 30-fold slower relaxation (depending on solvent) compared with guanine. A significant conformational change occurs at the S1 minimum, resulting in a 10-fold greater fluorescence quantum yield compared with guanine. The fluorescence quantum yield and S1 decay lifetime increase going from water to acetonitrile to propanol. The solvent-dependent results are supported by the quantum chemical calculations showing an increase in the energy barrier between the S1 minimum and the S1 /S0 conical intersection going from water to propanol. The longer lifetimes might make 5N7C G more photochemically active to adjacent nucleobases than guanine or other nucleobases within DNA.

Disclosing the Role of C4-Oxo Substitution in the Photochemistry of DNA and RNA Pyrimidine Monomers: Formation of Photoproducts from the Vibrationally Excited Ground State.

Oxo and amino substituted purines and pyrimidines have been suggested as protonucleobases participating in ancient pre-RNA forms. Considering electromagnetic radiation as a key environmental selection pressure on early Earth, the investigation of the photophysics of modified nucleobases is crucial to determine their viability as nucleobases' ancestors and to understand the factors that rule the photostability of natural nucleobases. In this Letter, we combine femtosecond transient absorption spectroscopy and quantum mechanical simulations to reveal the photochemistry of 4-pyrimidinone, a close relative of uracil. Irradiation of 4-pyrimidinone with ultraviolet radiation populates the S1(ππ*) state, which decays to the vibrationally excited ground state in a few hundred femtoseconds. Analysis of the postirradiated sample in water reveals the formation of a 6-hydroxy-5H-photohydrate and 3-(N-(iminomethyl)imino)propanoic acid as the primary photoproducts. 3-(N-(Iminomethyl)imino)propanoic acid originates from the hydrolysis of an unstable ketene species generated from the C4-N3 photofragmentation of the pyrimidine core.

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 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.