Strategic Design of Fluorescent Perylene-Modified Nucleic Acid Monomers: Position-, Phosphorylation-, and...

Synthetic fluorescent nucleotides (SFNs) have a wide variety of applications in biochemical tracking, imaging, and diagnostic assays. There are many SFNs in active development to enhance their fluorescence wavelengths, environmental sensitivity, photostability, and photochemical/photoswitchable properties. However, there are few systematic theoretical studies of their fluorescence properties. In this work, we apply excited-state QM/MM dynamics with TDDFT to nucleic acids tagged with perylene, which is particularly photostable, fluorescent, and bright (fluorescence quantum yield = 0.94) in isolation. We demonstrate that the overall structure, phosphorylation state, and linker type control electron transfer and fluorescence properties. A critical 90° dihedral angle between the perylene tag and nucleobase drives rapid quenching through charge transfer pathways, with positions 7 and 8 on guanine showing higher quenching propensity than position 2. Potential energy profiles reveal that the accessibility of the 90° base-tag geometries is critical for charge transfer, with the attachment position controlling this accessibility through steric and electronic effects. The presence of a phosphate group modulates this process by stabilizing excited states and reducing charge transfer rates. Additionally, ethynylene linkers maintain fluorescence through reduced angular dependence. The directionality of electron transfer stems primarily from the nucleic acid type, with guanine showing bidirectional transfer depending on the initial geometry, while adenine remains stable without significant charge transfer. These findings provide structural design principles for improved synthetic fluorescent nucleotides with tailored charge transfer characteristics.