Fluorescent light-up aptamers (FLAPs) have been used as label-free reporters in biosensing and bioimaging applications. Dapoxyl-binding aptamer DAP-10 is a recently reported DNA FLAP that holds potential as a scaffold for biosensor design due to its high affinity towards dapoxyl sulfonyl dyes, ability to bind other fluorogen emitting at a wide range of wavelength, and high fluorescence turn-on properties. The aptamer’s folding, the structure of its dye-binding site and the rational for its observed promiscuity in fluorogen binding is currently unknown, causing the design of biosensor constructs to be based on empirical principles and their reported performances possibly suboptimal. In this project we performed systematic mutagenesis of DAP-10 and employed fluorescence and circular dichroism (CD) spectroscopy to identify essential nucleotides involved in the dye binding and to shed light on the structure of the dye-aptamer complexes. Specifically, based on the CD data, potassium ion requirement and ability of the aptamer to enhance fluorescence of thioflavin T and N-methylmesoporphyrin IX, we confirmed folding of DAP10 into a G-quadruplex (GQ) motif stabilized by an adjacent stem structure. Interaction of the aptamer with either dapoxyl sulfonyl fluoride or a molecular rotor auramine O causes change from a predominantly parallel to a hybrid (3+1) GQ conformation. We speculate that the dye-binding site of DAP10 is formed at the junction between the GQ and duplex domains of the aptamer, with the dye involved in π-π stacking with the top G-tetrad of the GQ domain and the nucleotides of the stem to ensure continuity of coaxial stacking between the two domains, thus enabling fluorescence enhancement of the bound dyes.

Fluorogen binding by the dapoxyl binding aptamer DAP10 induced the aptamer folding into a mixed G-quadruplex/duplex architecture

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