Molecular analysis of RNA through hybridization with sequence-specific probes is challenging due to the intrinsic ability of RNA molecules to form stable secondary and tertiary structures. To overcome the energy barrier towards the probe-RNA complex formation, the probes are made of artificial nucleotides, which are more expensive than their natural counterparts and may still be inefficient. Here, we propose the use of a multicomponent probe based on an RNA-cleaving deoxyribozyme for the analysis of highly structured RNA targets. Efficient interrogation of two native RNA from Saccharomyces cerevisiae - a transfer RNA (tRNA) and 18S ribosomal RNA (rRNA) - was achieved at ambient temperature. We achieved detection limits of tRNA down to ~0.3 nM, which is 2 orders of magnitude lower than that previously reported for molecular beacon probes. Importantly, no probe annealing to the target was required, with the hybridization assay performed at 37o C. Excess of non-specific targets did not compromise the performance of the probe, and high interrogation efficiency was maintained by the probes even in complex matrices, such as cell lysate. A linear dynamic range of 0.1-150 nM tRNA was demonstrated. The probe can be adapted for differentiation of a single mismatch in the tRNA-probe complex. Therefore, this study opens a venue toward highly selective, sensitive, robust, and inexpensive assays for the interrogation of biological RNA.

中文翻译：

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在环境温度下用多组分脱氧核酶探针检测高度结构化的 RNA

由于 RNA 分子具有形成稳定二级和三级结构的内在能力，因此通过与序列特异性探针杂交对 RNA 进行分子分析具有挑战性。为了克服探针-RNA 复合物形成的能量障碍，探针由人工核苷酸制成，这比它们的天然对应物更昂贵，并且可能仍然效率低下。在这里，我们建议使用基于 RNA 切割脱氧核酶的多组分探针来分析高度结构化的 RNA 目标。在环境温度下实现了对来自酿酒酵母的两种天然 RNA - 转移 RNA (tRNA) 和 18S 核糖体 RNA (rRNA) 的有效询问。我们实现了低至 ~0.3 nM 的 tRNA 检测限，这比之前报告的分子信标探针低 2 个数量级。重要的是，不需要探针与靶标退火，杂交试验在 37o C 下进行。过量的非特异性靶标不会影响探针的性能，即使在复杂的基质中，探针也能保持高询问效率，例如作为细胞裂解液。证明了 0.1-150 nM tRNA 的线性动态范围。该探针可用于区分 tRNA 探针复合物中的单个错配。因此，这项研究为高选择性、灵敏、稳健且廉价的生物 RNA 检测开辟了道路。即使在复杂的基质（如细胞裂解液）中，探针也能保持高询问效率。证明了 0.1-150 nM tRNA 的线性动态范围。该探针可用于区分 tRNA 探针复合物中的单个错配。因此，这项研究为高选择性、灵敏、稳健且廉价的生物 RNA 检测开辟了道路。即使在复杂的基质（如细胞裂解液）中，探针也能保持高询问效率。证明了 0.1-150 nM tRNA 的线性动态范围。该探针可用于区分 tRNA 探针复合物中的单个错配。因此，这项研究为高选择性、灵敏、稳健且廉价的生物 RNA 检测开辟了道路。