In vitro selection of structure-switching, self-reporting aptamers

We describe an innovative selection approach to generate self-reporting aptamers (SRAs) capable of converting target-binding events into fluorescence readout without requiring additional modification, optimization, or the use of DNA helper strands. These aptamers contain a DNAzyme moiety that is initially maintained in an inactive conformation. Upon binding to their target, the aptamers undergo a structural switch that activates the DNAzyme, such that the binding event can be reported through significantly enhanced fluorescence produced by a specific stacking interaction between the active-conformation DNAzyme and a small molecule dye, N-methylmesoporphyrin IX. We demonstrate a purely in vitro selection-based approach for obtaining SRAs that function in both buffer and complex mixtures such as blood serum; after 15 rounds of selection with a structured DNA library, we were able to isolate SRAs that possess low nanomolar affinity and strong specificity for thrombin. Given ongoing progress in the engineering and characterization of functional DNA/RNA molecules, strategies such as ours have the potential to enable rapid, efficient, and economical isolation of nucleic acid molecules with diverse functionalities.