Split Spinach Aptamer for Highly Selective Recognition of DNA and RNA at Ambient Temperatures

Split spinach aptamer (SSA) probes for fluorescent analysis of nucleic acids were designed and tested. In SSA design, two RNA or RNA/DNA strands hybridized to a specific nucleic acid analyte and formed a binding site for low‐fluorescent 3,5‐difluoro‐4‐hydroxybenzylidene imidazolinone (DFHBI) dye, which resulted in up to a 270‐fold increase in fluorescence. The major advantage of the SSA over state‐of‐the art fluorescent probes is high selectivity: it produces only background fluorescence in the presence of a single‐base‐mismatched analyte, even at room temperature. SSA is therefore a promising tool for label‐free analysis of nucleic acids at ambient temperatures.