Forced Intercalation (FIT)-Aptamers

Aptamers are oligonucleotide sequences that can be evolved to bind to various analytes of interest. Here, we present a general design strategy that transduces an aptamer-target binding event into a fluorescence readout via the use of a viscosity-sensitive dye. Target binding to the aptamer leads to forced intercalation (FIT) of the dye between oligonucleotide base pairs, increasing its fluorescence by up to 20-fold. Specifically, we demonstrate that FIT-aptamers can report target presence through intramolecular conformational changes, sandwich assays, and target-templated reassociation of splitaptamers, showing that the most common aptamer-target binding modes can be coupled to a FIT-based readout. This strategy also can be used to detect the formation of a metallo-base pair within a duplexed strand and is therefore attractive for screening for metal-mediated base pairing events. Importantly, FIT-aptamers reduce false-positive signals typically associated with fluorophore-quencher based systems, quantitatively outperform FRET-based probes by providing up to 15-fold higher signal to background ratios, and allow rapid and highly sensitive target detection (nanomolar range) in complex media such as human serum. Taken together, FIT-aptamers are a new class of signaling aptamers which contain a single modification, yet can be used to detect a broad range of targets.