A Multiscale Computational and Experimental Study of TBA–Fluorescent Probes for Protein Sensing: Photobasicity Over Twisted Intramolecular Charge Transfer as a New Mechanism for Protein Induced Fluorescence Enhancement

Fluorescent probes are powerful tools for the detection of proteins in biomedical applications. However, the design of selectively active fluorescent probes is challenging due in part to difficulties predicting the functions of novel modifications, especially in different cellular environments. In the present study, a family of cyclic N-glycol-linked 4-formyl-aniline probes (denoted AnMeInd, AnMeBtz, and AnBtz), which have distinct thrombin binding affinities and fluorescent responses, were investigated at the T3 position of the thrombin binding aptamer (TBA) using TD-DFT calculations and classical and driven-adaptive bias molecular dynamics (MD) simulations. Classical MD and D-ABMD simulations corroborate the experimentally observed differences in thrombin binding affinities relative to canonical TBA, highlighting that neutral, shorter probes that lack an abundance of exocyclic sub-stituents are better accommodated in a thrombin binding pocket that is rich in aromatic and charged residues. TD-DFT suggests that cationic AnMeInd and AnMeBtz exhibit the same inherent tendency to undergo twisted intramolecular charge transfer (TICT). However, the methyl substituents of AnMeInd reduce probe contacts with the aptamer scaffold to enhance TICT in the unbound state, and adjust probe binding location in the thrombin hydrophobic pocket, which increases solvent shielding, probe rigidity, and TICT suppression upon target binding compared to AnMeBtz. Unlike the cationic ana-logues, combined computational and experimental data suggest the best AnBtz probe functions as a photobase. Although AnBtz undergoes protonation in the excited state in solvent exposed environ-ments (i.e., unbound TBA), which facilitates nonradiative decay, this pathway is suppressed upon thrombin binding due to reduced solvent accessibility, thereby enhancing fluorescence. These findings underscore the importance of tuning probe charge, linker length, exocyclic substituents, flexibility, and microenvironment in both the unbound and bound states to optimize fluorescence response. Our results provide a strategic foundation for designing high-performance fluorescent probes for biosensing and nucleic acid-based diagnostics.