Application of solid-phase DNA probe method with cleavage by deoxyribozyme for analysis of long non-coding RNAs.
Application of solid-phase DNA probe method with cleavage by deoxyribozyme for analysis of long non-coding RNAs.
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应用脱氧核酶切割固相 DNA 探针方法分析长链非编码 RNA。
DOI：
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
H. Hori
中科院分区：
文献类型：
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作者：
Shizuka Arakawa;Kohsuke Kamizaki;Y. Kuwana;Naruki Kataoka;C. Naoe;C. Takemoto;T. Yokogawa;H. Hori
固相DNA探针法是一种成熟的tRNA纯化技术。我们已经将这种方法应用于其他非编码RNA的纯化和分析。将用于纯化来自嗜热栖热菌的tRNAPhe、转移信使RNA（tmRNA）和16S rRNA的三个柱串联并进行纯化。从每个柱，tRNAPhe，tmRNA和16S rRNA可以在一个步骤中纯化。这是首次从T.嗜热菌和纯化证明固相DNA探针方法适用于非编码RNA，其以低于tRNA的量存在。此外，如果长的非编码RNA被位点特异性地切割并且该片段可以通过固相DNA探针方法纯化，则可以分析长的非编码RNA中的修饰核苷。因此，我们设计了脱氧核酶（DNAzyme）进行16S rRNA的位点特异性切割，检查最佳条件和纯化所得的RNA片段。互补DNA测序和质谱分析显示，纯化的RNA对应于16S rRNA的靶片段。因此，使用固相DNA探针方法的DNAzyme切割和纯化的组合可以是用于分析长非编码RNA中的修饰核苷的有用技术。
The solid-phase DNA probe method is a well-established technique for tRNA purification. We have applied this method for purification and analysis of other non-coding RNAs. Three columns for purification of tRNAPhe, transfer-messenger RNA (tmRNA) and 16S rRNA from Thermus thermophilus were connected in tandem and purifications were performed. From each column, tRNAPhe, tmRNA and 16S rRNA could be purified in a single step. This is the first report of purification of native tmRNA from T. thermophilus and the purification demonstrates that the solid-phase DNA probe method is applicable to non-coding RNA, which is present in lower amounts than tRNA. Furthermore, if a long non-coding RNA is cleaved site-specifically and the fragment can be purified by the solid-phase DNA probe method, modified nucleosides in the long non-coding RNA can be analyzed. Therefore, we designed a deoxyribozyme (DNAzyme) to perform site-specific cleavage of 16S rRNA, examined optimum conditions and purified the resulting RNA fragment. Sequencing of complimentary DNA and mass spectrometric analysis revealed that the purified RNA corresponded to the targeted fragment of 16S rRNA. Thus, the combination of DNAzyme cleavage and purification using solid-phase DNA probe methodology can be a useful technique for analysis of modified nucleosides in long non-coding RNAs.
影响因子：
4.5
作者：
Jackman, JE;Montange, RK;Phizicky, EM
通讯作者：
Phizicky, EM