On-Strand Knoevenagel Insertion of a Hemicyanine Molecular Rotor Loop Residue for Turn-On Fluorescence Detection of Pb-Induced G‑Quadruplex Rigidity

We demonstrate the ability to distinguish Pb2+ from K+ within the central cavity of the antiparallel G-quadruplex (GQ) DNA produced by the thrombin binding aptamer (TBA) using an internal molecular rotor fluorescent probe. An indole–aldehyde containing an acyclic N-glycol group was first employed in the on-strand Knoevenagel condensation with five different heterocyclic quaternary cationic acceptors to assess the molecular rotor character of the resulting cyanine–styryl dyes within duplex DNA. An indole–pyridinium (4PI) nucleobase surrogate displayed the greatest turn-on emission response to duplex formation and was thus inserted into the loop residues of TBA to monitor GQ-folding in the presence of Pb2+ versus K+. TBA-4PI exhibits turn-on emission upon Pb2+-binding with a brightness (ε·Φfl) of 9000 cm–1 M–1 compared to K+-binding (ε·Φfl ∼ 2000 cm–1 M–1) due to Pb2+-induced GQ rigidity with 4PI-G-tetrad stacking interactions. The Pb2+-bound TBA-4PI GQ also provides energy-transfer (ET) fluorescence with a diagnostic excitation at 310 nm for distinguishing Pb2+ from K+ within the antiparallel GQ. The TBA-4PI GQ affords the desired turn-on fluorescence response for detecting Pb2+ ions with an apparent dissociation constant (K d) of 63 nM and a limit of detection (LOD) of 19 nM in an aqueous buffer. It can also distinguish Pb2+ (230 nM) from K+ (1.5 mM, 6500-fold excess) in an antiparallel GQ recognition motif without topology twitching.