Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan.
Department of Chemistry, Faculty of Science, Tokyo University of Science, Tokyo 162-8601, Japan.
RNA. 2019 May;25(5):590-599. doi: 10.1261/rna.069062.118. Epub 2019 Feb 11.
RNAs play essential roles in various cellular processes and can be used as biomarkers. Hence, it is important to detect endogenous RNA for understanding diverse cellular functions and diagnosing diseases. To construct a low-cost and easy-to-use RNA detection probe, a chemically unmodified RNA aptamer that binds to a pro-fluorophore to increase its fluorescence is desirable. Here, we focused on Broccoli, a superior variant of Spinach, which is a well-known fluorescent RNA aptamer that binds to DFHBI-1T and emits green fluorescence. We experimentally characterized Broccoli and predicted that it forms a G-quadruplex-based DFHBI-1T recognition region sandwiched between two stems. Based on this, we designed a Broccoli-based RNA detection probe (BRD probe) composed of a sequence of destabilized Broccoli fused with complementary sequences against target RNA. The resulting probe with its target RNA formed a stable three-way junction, named the MT2 three-way junction, which contributed to efficient refolding of the Broccoli structure and allowed for programmable RNA detection with high signal-to-noise ratio and sensitivity. Interestingly, the MT2 three-way junction also could be applied to probe construction of a truncated form of Spinach (Baby Spinach). The BRD and Baby Spinach-based RNA detection probes (BSRD probe) exhibited up to 48- and 140-fold fluorescence enhancements in the presence of their target RNAs and detected small amounts of target RNA that were as low as 160 and 5 nM, respectively. Thus, we experimentally characterized the higher order structure of Broccoli and developed structure-switching aptamer probes for highly sensitive, programmable, RNA detection using an MT2 three-way junction.
RNAs 在各种细胞过程中发挥着重要作用，并且可以用作生物标志物。因此，检测内源性 RNA 对于理解多种细胞功能和诊断疾病非常重要。为了构建低成本且易于使用的 RNA 检测探针，需要一种化学修饰的 RNA 适体，该适体可以与前荧光团结合以增加其荧光。在这里，我们专注于 Broccoli，它是 Spinach 的一个优越变体，是一种众所周知的荧光 RNA 适体，可与 DFHBI-1T 结合并发出绿色荧光。我们对 Broccoli 进行了实验表征，并预测它形成了基于 G-四链体的 DFHBI-1T 识别区域，夹在两个茎之间。在此基础上，我们设计了一种基于 Broccoli 的 RNA 检测探针 (BRD 探针)，由与靶 RNA 互补的不稳定 Broccoli 序列组成。所得探针与其靶 RNA 形成了稳定的三链结，称为 MT2 三链结，有助于 Broccoli 结构的有效重折叠，并允许使用高信噪比和灵敏度进行可编程 RNA 检测。有趣的是，MT2 三链结也可应用于截短形式的 Spinach（Baby Spinach）的探针构建。BRD 和 Baby Spinach 基 RNA 检测探针 (BSRD 探针) 在存在其靶 RNA 时，荧光增强分别高达 48 倍和 140 倍，并分别检测到低至 160 和 5 nM 的少量靶 RNA。因此，我们对 Broccoli 的高级结构进行了实验表征，并开发了基于结构切换的适体探针，用于使用 MT2 三链结进行高灵敏度、可编程的 RNA 检测。
