Benzimidazole Modification of DNA Aptamers for Ultra Responsive Protein-Induced Fluorescence Enhancement
Year: 2026
Type: preprint
Abstract: Post-SELEX incorporation of environmentally responsive fluorescent nucleobase surrogates provides a powerful strategy for converting DNA aptamers into protein aptasensors, yet achieving large protein-induced fluorescence enhancement (PIFE) while preserving target affinity remains a major challenge.Here, we introduce a photobasic 4-(1H-benzo[d]imidazol-2-yl)-N,N-dimethylaniline (AnIm) nucleobase surrogate that enables ultraresponsive PIFE through site-specific incorporation into DNA aptamers.As a model system, AnIm was incorporated into two thrombin-binding aptamers that recognize distinct protein exosites: the 15-mer TBA15 and the 27-mer HD22.The modified aptamers retained high thrombin affinity (Kd = 85-167 nM).Within HD22, site-dependent fluorescence enhancements of 70-108-fold (Irel) were observed, affording a limit of detection (LOD) of 280 pM in PBS and 3.8 nM in 5% human serum.To our knowledge, this >100-fold fluorescence turn-on represents the largest PIFE response reported for a fluorescent nucleobase surrogate.Spectroscopic studies reveal that the exceptional sensitivity arises from a protein-induced pKa shift coupled with suppression of a twisted intramolecular charge-transfer (TICT) state.Protein binding shifts AnIm from its protonated (NH⁺) ground-state toward the neutral (N) form, reducing background fluorescence, while excited-state protonation generates a fluorescent molecular rotor whose emission is enhanced within the rigid protein microenvironment.These findings establish photobasic nucleobase surrogates as a general strategy for post-SELEX fluorescent aptamer engineering for ultra-responsive PIFE-based biosensing.
Source: ChemRxiv
Institutions: University of Guelph, University of Lethbridge
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7213303079