Programmable RNA detection with a fluorescent RNA aptamer using optimized three-way junction formation
Programmable RNA detection with a fluorescent RNA aptamer using optimized three-way junction formation
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DOI：
10.1261/rna.069062.118
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发表时间：
2019-05-01
期刊：
影响因子：
4.5
通讯作者：
Yoshimoto, Keitaro
中科院分区：
文献类型：
--
作者：
Furuhata, Yuichi;Kobayashi, Mizuki;Yoshimoto, Keitaro
RNA在各种细胞过程中发挥重要作用，可用作生物标志物。因此，检测内源性RNA对于了解多种细胞功能和诊断疾病具有重要意义。为了构建低成本且易于使用的RNA检测探针，需要结合前荧光团以增加其荧光的化学未修饰的RNA适体。在这里，我们专注于花椰菜，菠菜的一种上级变体，它是一种众所周知的荧光RNA适体，与DFHBI-1 T结合并发出绿色荧光。我们的实验特征花椰菜和预测，它形成了一个G-四链体为基础的DFHBI-1 T识别区夹在两个茎。基于此，我们设计了一种基于花椰菜的RNA检测探针（BRD探针），该探针由不稳定的花椰菜序列与针对靶RNA的互补序列融合而成。所得探针与其靶RNA形成稳定的三向连接，称为MT 2三向连接，这有助于花椰菜结构的有效重折叠，并允许具有高信噪比和灵敏度的可编程RNA检测。有趣的是，MT 2三向连接也可以应用于构建截短形式的菠菜（菠菜宝宝）。基于BRD和小菠菜的RNA检测探针（BSRD探针）在其靶RNA存在下表现出高达48倍和140倍的荧光增强，并且分别检测到低至160和5 nM的少量靶RNA。因此，我们的实验特征的西兰花的高阶结构和开发的结构转换适体探针的高灵敏度，可编程的，RNA检测使用MT 2的三通接头。
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.