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PreprintCC BYChemRxiv

Benzimidazole Modification of DNA Aptamers for Ultra Responsive Protein-Induced Fluorescence Enhancement

  • Matteo Pavlovic1,2
  • Austin Pounder3,4
  • Naomi Varnovitski1,2
  • Stacey D. Wetmore3,4
  • Richard A. Manderville1,2
4 affiliations
  1. 1Department of Chemistry and Toxicology
  2. 2University of Guelph
  3. 3Department of Chemistry and Biochemistry
  4. 4University of Lethbridge
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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.

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Cite

BibTeXRIS
BibTeX
@misc{pavlovic2026benzimidazole,
  title = {{Benzimidazole Modification of DNA Aptamers for Ultra Responsive Protein-Induced Fluorescence Enhancement}},
  author = {Matteo Pavlovic and Austin Pounder and Naomi Varnovitski and Stacey D. Wetmore and Richard A. Manderville},
  howpublished = {ChemRxiv},
  year = {2026},
  month = sep,
  doi = {10.26434/chemrxiv.15008836/v1},
  url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15008836/v1},
  note = {CC BY},
}
RIS
TY  - UNPB
TI  - Benzimidazole Modification of DNA Aptamers for Ultra Responsive Protein-Induced Fluorescence Enhancement
AU  - Matteo Pavlovic
AU  - Austin Pounder
AU  - Naomi Varnovitski
AU  - Stacey D. Wetmore
AU  - Richard A. Manderville
PY  - 2026
DA  - 2026/09/15
PB  - ChemRxiv
DO  - 10.26434/chemrxiv.15008836/v1
UR  - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15008836/v1
AB  - 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.
ER  -