Department of Pharmacology, Weill-Cornell Medical College, Cornell University, New York, New York 10065, United States.
Tri-Institutional PhD Program in Chemical Biology, Weill Cornell Medicine, Memorial Sloan Kettering Cancer Center, The Rockefeller University, New York, New York 10065, United States.
ACS Chem Biol. 2022 Apr 15;17(4):840-853. doi: 10.1021/acschembio.1c00893. Epub 2022 Mar 26.
RNA detection is important in diverse diagnostic and analytical applications. RNAs can be rapidly detected using molecular beacons, which fluoresce upon hybridizing to a target RNA but require oligonucleotides with complex fluorescent dye and quencher conjugations. Here, we describe a simplified method for rapid fluorescence detection of a target RNA using simple unmodified DNA oligonucleotides. To detect RNA, we developed Lettuce, a fluorogenic DNA aptamer that binds and activates the fluorescence of DFHBI-1T, an otherwise nonfluorescent molecule that resembles the chromophore found in green fluorescent protein. Lettuce was selected from a randomized DNA library based on binding to DFHBI-agarose. We further show that Lettuce can be split into two separate oligonucleotide components, which are nonfluorescent on their own but become fluorescent when their proximity is induced by a target RNA. We designed several pairs of split Lettuce fragments that contain an additional 15-20 nucleotides that are complementary to adjacent regions of the SARS-CoV-2 RNA, resulting in Lettuce fluorescence only in the presence of the viral RNA. Overall, these studies describe a simplified RNA detection approach using fully unmodified DNA oligonucleotides that reconstitute the Lettuce aptamer templated by RNA.
RNA 检测在各种诊断和分析应用中都很重要。可以使用分子信标快速检测 RNA，当与靶 RNA 杂交时，分子信标会发出荧光，但需要具有复杂荧光染料和淬灭剂结合的寡核苷酸。在这里，我们描述了一种使用简单未修饰的 DNA 寡核苷酸快速检测靶 RNA 的简化方法。为了检测 RNA，我们开发了 Lettuce，它是一种荧光 DNA 适体，可与 DFHBI-1T 结合并激活其荧光，DFHBI-1T 是一种原本没有荧光的分子，类似于绿色荧光蛋白中的发色团。Lettuce 是根据与 DFHBI-琼脂糖的结合从随机化的 DNA 文库中选择的。我们进一步表明，Lettuce 可以分裂成两个单独的寡核苷酸组件，它们本身没有荧光，但当其接近由靶 RNA 诱导时，它们会变得荧光。我们设计了几对分裂的 Lettuce 片段，它们包含另外 15-20 个与 SARS-CoV-2 RNA 相邻区域互补的核苷酸，只有在存在病毒 RNA 的情况下，才会产生 Lettuce 荧光。总的来说，这些研究描述了一种使用完全未修饰的 DNA 寡核苷酸进行简化 RNA 检测的方法，该方法重新构成了由 RNA 模板化的 Lettuce 适体。
