The simple 5-furyl-2'-deoxyuridine ((Fur)dU) nucleobase exhibits dual probing characteristics displaying emissive sensitivity to changes in microenvironment polarity and to changes in solvent rigidity due to its molecular rotor character. Here, we demonstrate its ability to define the microenvironment of the various thymidine (T) loop residues within the thrombin binding aptamer (TBA) upon antiparallel G-quadruplex (GQ) folding and thrombin binding. The emissive sensitivity of the (Fur)dU probe to microenvironment polarity provides a diagnostic handle to distinguish T bases that are solvent-exposed within the GQ structure compared with probe location in the apolar duplex. Its molecular rotor properties then provide a turn-on fluorescent switch to identify which T residues within the GQ bind specifically to the protein target (thrombin). The fluorescence sensing characteristics of (Fur)dU make it an attractive tool for mapping aptamer-protein interactions at the nucleoside level for further development of modified aptamers for a wide range of diagnostic and therapeutic applications.

We demonstrate the ability to distinguish Pb 2+ 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 Pb 2+ versus K + . TBA-4PI exhibits turn-on emission upon Pb 2+ -binding with a brightness (ε•Φ fl ) of 9000 cm −1 M −1 compared to K +binding (ε•Φ fl ∼ 2000 cm −1 M −1 ) due to Pb 2+ -induced GQ rigidity with 4PI-Gtetrad stacking interactions. The Pb 2+ -bound TBA-4PI GQ also provides energytransfer (ET) fluorescence with a diagnostic excitation at 310 nm for distinguishing Pb 2+ from K + within the antiparallel GQ. The TBA-4PI GQ affords the desired turn-on fluorescence response for detecting Pb 2+ 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 Pb 2+ (230 nM) from K + (1.5 mM, 6500-fold excess) in an antiparallel GQ recognition motif without topology twitching.

Chemically modified aptamers have the opportunity to increase aptamer target binding affinity and provide structure-activity relationships to enhance our understanding of molecular target recognition by the aptamer fold. In the current study, 8-aryl-2'-deoxyguanosine nucleobases have been inserted into the G-tetrad and central TGT loop of the thrombin binding aptamer (TBA) to determine their impact on antiparallel G-quadruplex (GQ) folding and thrombin binding affinity. The aryl groups attached to the dG nucleobase vary greatly in aryl ring size and impact on GQ stability (∼20 °C change in GQ thermal melting (Tm) values) and thrombin binding affinity (17-fold variation in dissociation constant (Kd)). At G8 of the central TGT loop that is distal from the aptamer recognition site, the probes producing the most stable GQ structure exhibited the strongest thrombin binding affinity. However, within the G-tetrad, changes to the electron density of the dG component within the modified nucleobase can diminish thrombin binding affinity. Detailed molecular dynamics (MD) simulations on the modified TBA (mTBA) and mTBA-protein complexes demonstrate how the internal 8-aryl-dG modification can manipulate the interactions between the DNA nucleobases and the amino acid residues of thrombin. These results highlight the potential of internal fluorescent nuclobase analogs (FBAs) to broaden design options for aptasensor development.

Fluorescent nucleobases represent an important class of molecular reporters of nucleic acid interactions. In this work, the advantages of utilizing a noncanonical fluorescent nucleobase surrogate for monitoring thrombin binding by the 15-mer thrombin binding aptamer (TBA) is presented. TBA folds into an antiparallel G-quadruplex (GQ) with loop thymidine (T) residues interacting directly with the protein in the thrombin−TBA complex. In the free GQ, T3 is solventexposed and does not form canonical base-pairs within the antiparallel GQ motif. Upon thrombin binding, T3 interacts directly with a hydrophobic protein binding pocket. Replacing T3 with a cyanine-indole-quinolinium (4QI) hemicyanine dye tethered to an acyclic 1,2-propanediol linker is shown to have minimal impact on GQ stability and structure with the internal 4QI displaying a 40-fold increase in emission intensity at 586 nm (excitation 508 nm) compared to the free dye in solution. Molecular dynamics (MD) simulations demonstrate that the 4QI label π-stacks with T4 and T13 within the antiparallel GQ fold, which is supported by strong energy transfer (ET) fluorescence from the GQ (donor) to the 4QI label (acceptor). Thrombin binding to 4QI-TBA diminishes π-stacking interactions between 4QI and the GQ structure to cause a turn-off emission intensity response with an apparent dissociation constant (K d ) of 650 nM and a limit of detection (LoD) of 150 nM. These features highlight the utility of internal noncanonical fluorescent surrogates for monitoring protein binding by GQfolding aptamers in the absence of DNA topology switching.

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 N-methyl-4-bromoaniline (4-Br-An) containing an acyclic N-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 singlestrand 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.