Strategic Design of Fluorescent Perylene-Modified Nucleic Acid Monomers: Position‑, Phosphorylation‑, and Linker-Dependent Control of Electron Transfer

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.