Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
Anal Biochem. 2010 May 1;400(1):103-9. doi: 10.1016/j.ab.2010.01.015. Epub 2010 Jan 20.
Aptamers are short single-stranded DNA or RNA sequences that are selected in vitro based on their high affinity to a target molecule. Dye-binding aptamers are promising tools for real-time detection of not only DNA or RNA sequences but also proteins of interest both in vitro and in vivo. In this study, we aimed to isolate an RNA aptamer to Cy3, a widely used, membrane-permeant, and nontoxic fluorescent cyanine dye. Extensive selection of affinity RNA molecules to Cy3 yielded a unique sequence aptamer named Cy3_apt. The selected Cy3_apt was 83 nucleotides long and successfully shortened to 49 nucleotides long with increased affinity to Cy3 by multiple base changes. The shortest Cy3_apt is composed of two separate hairpin modules that are required for the affinity to Cy3 as monitored by the surface plasmon resonance (SPR) assay. Also, the fluorescence of Cy3 increased on binding to Cy3_apt. The two modules of Cy3_apt, when detached from each other, functioned as a binary aptamer probe. We demonstrate that the binary Cy3_apt probe is applicable to the detection of target oligonucleotides or RNA-RNA interaction by tagging with target sequences. This binary probe consists of two folded modules, referred to as a folded binary probe.
适体是经过体外筛选得到的短单链 DNA 或 RNA 序列，对靶分子具有高亲和力。染料结合适体是一种很有前途的工具，不仅可用于实时检测体外和体内的 DNA 或 RNA 序列，还可用于实时检测感兴趣的蛋白质。在这项研究中，我们旨在分离与 Cy3 结合的 RNA 适体，Cy3 是一种广泛使用的、膜通透性的、非毒性的荧光菁染料。对 Cy3 进行了广泛的亲和力 RNA 分子选择，得到了一个独特的序列适体，命名为 Cy3_apt。所选的 Cy3_apt 长 83 个核苷酸，通过多次碱基变化成功缩短至 49 个核苷酸，与 Cy3 的亲和力增加。最短的 Cy3_apt 由两个单独的发夹模块组成，这两个模块是通过表面等离子体共振 (SPR) 分析监测到的与 Cy3 结合的必需条件。此外，Cy3 的荧光在与 Cy3_apt 结合时增强。Cy3_apt 的两个模块彼此分离时，作为二元适体探针发挥作用。我们证明，二元 Cy3_apt 探针可通过标记靶序列应用于靶寡核苷酸或 RNA-RNA 相互作用的检测。该二元探针由两个折叠模块组成，称为折叠二元探针。
