G-Quadruplexes (GQs) serve as popular recognition elements for DNA aptasensors and are incorporated into a DNA nanodevice capable of controlled conformational changes to activate a sensing mechanism. Herein we highlight the utility of a GQ−GQ nanodevice fueled by GQ-specific ligands as a label-free aptasensor detection strategy. The concept was first illustrated utilizing the prototypical polymorphic human telomeric repeat sequence (H-Telo22, d[AG3(T2AG3)3]) that can undergo ligand-induced topology changes between antiparallel, parallel, or hybrid GQ structures. The H-Telo22−ligand interactions served as a model of the GQ−GQ nanodevice. The utility of the device in a real aptasensor platform was then highlighted utilizing the ochratoxin A (OTA) binding aptamer (OTABA) that folds into an antiparallel GQ in the absence and presence of target OTA. Three cationic fluorogenic ligands served as GQ-specific light-up probes and as potential fuel for the GQ−GQ nanodevice by producing an inactive GQ topology (parallel or hybrid) of OTABA. Our findings demonstrate efficient OTAmediated dye displacement with excellent emission sensitivity for OTA detection when the fluorogenic dyes induce a topology change in OTABA (parallel or hybrid). However, when the fluorogenic dye fails to induce a conformational change in the antiparallel fold of OTABA, subsequent additions of OTA to the aptamer−dye complex results in poor dye displacement with weak emission response for OTA detection. These results are the first to exemplify a ligand-induced GQ−GQ nanodevice as an aptasensor mechanism and demonstrate diagnostic applications for topology-specific GQ binders.

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.

Acceptor aryl groups at the 8-position of 2′-deoxyguanosine (dG) generate visibly emissive 8aryldG probes, which provide viscosity-sensing applications within oligonucleotides.

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.

Ochratoxin A (OTA) is an intrinsically fluorescent phenolic mycotoxin that contaminates a wide range of food products and is a serious health threat to animals and humans. An OTA binding aptamer (OTABA) that folds into an antiparallel G-quadruplex (GQ) in the absence and presence of target OTA has been incorporated into a vast variety of aptasensor platforms for OTA detection. The development of a simple, aptamer-based approach would allow for detection of the toxin without the use of complex analytical instrumentation, which has been the gold standard for OTA detection thus far. However, to date, none of the aptasensor platforms have utilized the natural fluorescence of the phenolic toxin for detection. Herein, we report that OTA binding to OTABA involves π-stacking interactions that lead to GQ-to-toxin energy transfer (ET), which affords a “turn-on” fluorescence self-signaling platform in which the emission of the aptamer–target complex is enhanced in comparison to the free toxin alone. Selective excitation of the GQ–OTA complex at 256 nm leads to a 4-fold enhancement in OTA fluorescence. The GQ–OTA ET detection platform boasts a limit of detection ∼2 ng/mL, which is comparable to a previously demonstrated fluorescence resonance energy transfer immunoassay platform for OTA detection, and displays excellent OTA selectivity and recovery from red wine samples.