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.

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.

High bacterial loads within chronic wounds increase the risk of infection and complication. Detection and localization of bacterial loads through point‐of‐care fluorescence (FL) imaging can objectively inform and support bacterial treatment decisions. This single time‐point, retrospective analysis describes the treatment decisions made on 1000 chronic wounds (DFUs, VLUs, PIs, surgical wounds, burns, and others) at 211 wound‐care facilities across 36 US states. Clinical assessment findings and treatment plans derived from them, as well as subsequent FL‐imaging (MolecuLight®) findings and any associated treatment plan changes, were recorded for analysis. FL signals indicating elevated bacterial loads were observed in 701 wounds (70.8%), while only 293 (29.6%) showed signs/symptoms of infection. After FL‐imaging, treatment plans changed in 528 wounds as follows: more extensive debridement (18.7%), more extensive hygiene (17.2%), FL‐targeted debridement (17.2%), new topical therapies (10.1%), new systemic antibiotic prescriptions (9.0%), FL‐guided sampling for microbiological analysis (6.2%), and changes in dressing selection (3.2%). These real‐world findings of asymptomatic bacterial load/biofilm incidence, and of the frequent treatment plan changes post‐imaging, are in accordance with clinical trial findings using this technology. These data, from a range of wound types, facilities, and clinician skill sets, suggest that point‐of‐care FL‐imaging information improves bacterial infection management.