Thieno[3,2‑b]thiophene for the Construction of Far-Red Molecular Rotor Hemicyanines as High-Affinity DNA Aptamer Fluorogenic Reporters

• Published in: Analytical chemistry (Washington) 2024-10, Vol.96 (41), p.16252-16259
• Main Authors: Johnson, Ryan E., Pounder, Austin, van der Zalm, Joshua, Chen, Aicheng, Bell, Ian J., Van Raay, Terence J., Wetmore, Stacey D., Manderville, Richard A.
• Format: Article
• Language: English
• Subjects: Aptamers; Aptamers, Nucleotide - chemistry; Binding; Carbocyanines - chemistry; Charge transfer; Deoxyribonucleic acid; DNA; DNA probes; Electrochemistry; Fluorescence; Fluorescent Dyes - chemistry; Humans; Molecular Structure; NMR; Nuclear magnetic resonance; Nucleic acids; Quinine; Rotors; Thiophenes - chemistry
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.4c03151

The construction of far-red fluorescent molecular rotors (FMRs) is an imperative task for developing nucleic acid stains that have superior compatibility with cellular systems and complex matrices. A typical strategy relies on the methine extension of asymmetric cyanines, which unfortunately fails to produce sensitive rotor character. To break free from this paradigm, we have synthesized far-red hemicyanines using a dimethylamino thieno­[3,2-b]­thiophene donor. The resultant probes, designated as ATh2Ind and ATh2Btz, possess excitation maxima (λmax) of >600 nm and have been rigorously characterized by NMR, electrochemistry, and computational methods. The dyes possess alternating charge patterns like indodicarbocyanine (Cy5), but with twisted intramolecular charge transfer (TICT) rotational barriers at 60°, akin to the classical FMR thiazole orange (TO1). ATh2Btz also displays cyanine characteristics, enhancing its response upon binding to nucleic acids and allowing for efficient staining of cellular nuclei. When binding to the DNA aptamer for quinine (MN4), ATh2Btz exhibits a K d of 17 nM, a 660-fold light-up response, brightness (Φfl x εmax) of ∼37,000 M–1cm–1, and λex/λem of 655/677 nm. The resulting far-red DNA-based MN4-ATh2Btz platform has been termed “pomegranate.”