Small molecule aptamer assays based on fluorescence anisotropy signal-enhancer oligonucleotides

• A novel small molecule fluorescence anisotropy assay was described.
• It relied on enhanced fluctuations of segmental motion dynamics of the aptamer tracer.
• Assay response was obtained with both pre-structured and unstructured aptamers.
• Assay response was obtained with dyes of different photochemical properties.
• Simultaneous multicolour fluorescence anisotropy detection was accomplished.

Herein, we design novel fluorescence anisotropy (FA) aptamer sensing platforms dedicated to small molecule detection. The assay strategy relied on enhanced fluctuations of segmental motion dynamics of the aptamer tracer mediated by an unlabelled, partially complementary oligonucleotide. The signal-enhancer oligonucleotide (SEO) essentially served as a free probe fraction revealer. By targeting specific regions of the signalling functional nucleic acid, the SEO binding to the unbound aptamer triggered perturbations of both the internal DNA flexibility and the localized dye environment upon the free probe to duplex structure transition. This potentiating effect determined increased FA variations between the duplex and target bound states of the aptameric probe. FA assay responses were obtained with both pre-structured (adenosine) and unstructured (tyrosinamide) aptamers and with dyes of different photochemical properties (fluorescein and texas red). The multiplexed analysis ability was further demonstrated through the simultaneous multicolour detection of the two small targets. The FA method appears to be especially simple, sensitive and widely applicable.