Sando Shinsuke, Narita Atsushi, Aoyama Yasuhiro
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Chembiochem. 2007 Oct 15;8(15):1795-803. doi: 10.1002/cbic.200700325.
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 tBu 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.
我们设计了一种策略，基于对传统DNA染色染料进行下调修饰以抑制其与原始双链DNA靶标的亲和力，从而生成一种发光的荧光团-适配体对，随后重新筛选能够与修饰后的染料结合的适配体。按照这一思路，我们制备了一种对微极性敏感的Hoechst衍生物，其具有两个叔丁基，对通常富含AT的双链DNA靶标亲和力较低。从随机文库中体外筛选出的DNA适配体可作为触发剂，增强原本无荧光的Hoechst衍生物的荧光，缩短后的25聚体序列显示出显著的增强效果（发光）。将25聚体序列拆分为二元适配体探针，这样我们就能够以单核苷酸分辨率检测靶核酸序列，使用未修饰的DNA作为探针即可实现。
