Interrogation of highly structured RNA with multicomponent deoxyribozyme probes at ambient temperatures.
Interrogation of highly structured RNA with multicomponent deoxyribozyme probes at ambient temperatures.
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DOI：
10.1261/rna.074864.120
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发表时间：
2020-12
期刊：
影响因子：
--
通讯作者：
Gerasimova YV
中科院分区：
文献类型：
--
作者：
Reed AJ;Sapia RJ;Dowis C;Solarez S;Gerasimova YV
由于RNA分子形成稳定的二级和三级结构的固有能力，通过与序列特异性探针杂交进行RNA的分子分析是具有挑战性的。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，人工核苷酸比其天然对应物更昂贵，并且可能仍然效率低下。在这里，我们建议使用的多组分探针的基础上的RNA切割脱氧核酶的高度结构化的RNA目标的分析。在环境温度下，实现了对来自酵母的两种天然RNA-转移RNA（tRNA）和18 S核糖体RNA（rRNA）的有效询问。我们实现了检测限的tRNA下降到10.3 nM，这是两个数量级低于以前报道的分子信标探针。重要的是，在37°C下进行杂交测定时，不需要探针与靶标退火。过量的非特异性靶并不损害探针的性能，并且即使在复杂的基质如细胞裂解物中，探针也能保持高的询问效率。证明了0.3-150 nM tRNA的线性动态范围。该探针可适用于区分tRNA-探针复合物中的单个错配。因此，这项研究为高选择性、灵敏度、稳健性和廉价的生物RNA检测开辟了一条道路。
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 toward 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 two 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 37°C. Excess of nonspecific 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 0.3–150 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.
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影响因子：
16.6
作者：
Ranasinghe RT;Challand MR;Ganzinger KA;Lewis BW;Softley C;Schmied WH;Horrocks MH;Shivji N;Chin JW;Spencer J;Klenerman D
通讯作者：
Klenerman D
影响因子：
3.2
作者：
Gerasimova, Yulia V.;Cornett, Evan;Kolpashchikov, Dmitry M.
通讯作者：
Kolpashchikov, Dmitry M.
影响因子：
16.6
作者：
Grimes, Jeffrey;Gerasimova, Yulia V.;Kolpashchikov, Dmitry M.
通讯作者：
Kolpashchikov, Dmitry M.
影响因子：
4.5
作者：
Gentry, Riley C.;Childs, Jared J.;Koculi, Eda
通讯作者：
Koculi, Eda
具有L-DNA茎的发夹核酸探针的优质结构稳定性和选择性。
影响因子：
14.9
作者：
Kim, Youngmi;Yang, Chaoyong James;Tan, Weihong
通讯作者：
Tan, Weihong