Screening Internal Donor-Acceptor Biaryl Nucleobase Surrogates for Turn-On Fluorescence Affords an Aniline-Carboxythiophene Probe for Protein Detection by G-Quadruplex DNA.

Donor-acceptor biaryls serve as microenvironment fluorescent sensors with highly quenched intramolecular charge transfer (ICT) emission in polar protic solvents that turns on in aprotic media. In DNA, canonical donor-acceptor fluorescent base analogs can be prepared through on-strand Suzuki-Miyaura cross-coupling reactions involving 8-bromo-2'-deoxyguanosine (8-Br-dG) with an acceptor aryboronic acid. Herein, we demonstrate that replacement of 8-Br-dG with -methyl-4-bromoaniline (4-Br-An) containing an acyclic -glycol group can be employed in the on-strand Suzuki-Miyaura reaction to afford new donor-acceptor biaryl nucleobase surrogates with a 40-fold increase in emission intensity for fluorescent readout within single-strand oligonucleotides. Screening the best acceptor for turn-on fluorescence upon duplex formation afforded the carboxythiophene derivative [COOTh]An with a 7.4-fold emission intensity increase upon formation of a single-bulged duplex (-1) with the surrogate occupying a pyrimidine-flanked bulge. Insertion of the [COOTh]An surrogate into the lateral TT loops produced by the antiparallel G-quadruplex (GQ) of the thrombin binding aptamer (TBA) afforded a 4.1-fold increase in probe fluorescence that was accompanied by a 20 nm wavelength shift to the blue upon thrombin binding. The modified TBA afforded a limit of detection of 129 nM for thrombin and displayed virtually no emission response to off-target proteins. The fluorescence response of [COOTh]An to thrombin binding highlights the utility of the thienyl-aniline moiety for monitoring DNA-protein interactions.