Light‐Up Hoechst–DNA Aptamer Pair: Generation of an Aptamer‐Selective Fluorophore from a Conventional DNA‐Staining Dye

Abstract We have designed a strategy to generate a light‐up fluorophore–aptamer pair based on a down‐modification of a conventional DNA‐staining dye to suppress its affinity to the original dsDNA targets, followed by reselection of aptamers that would bind to the modified dye. Following this line, we prepared a micropolarity‐sensitive Hoechst derivative possessing two t Bu groups with low affinity to the usual AT‐rich dsDNA targets. DNA aptamers selected in vitro from a random pool worked as triggers to enhance the fluorescence of an otherwise nonfluorescent Hoechst derivative, and the shortened 25‐mer sequence showed remarkable enhancement (light‐up). The 25‐mer sequence was split into binary aptamer probes, thus enabling us to detect a target nucleic acid sequence with a single‐nucleotide resolution by use of unmodified DNA as a probe.