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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.more » « lessFree, publicly-accessible full text available April 21, 2026
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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.more » « less
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