Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
J Biochem. 2020 Sep 1;168(3):273-283. doi: 10.1093/jb/mvaa048.
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 analysed. 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.
固相 DNA 探针法是一种成熟的 tRNA 纯化技术。我们已经将该方法应用于其他非编码 RNA 的纯化和分析。从嗜热栖热菌中纯化苯丙氨酸 tRNA、转移信使 RNA（tmRNA）和 16S rRNA 的三个柱串联在一起，并进行了纯化。从每个柱中，可以一步纯化苯丙氨酸 tRNA、tmRNA 和 16S rRNA。这是首次从嗜热栖热菌中纯化天然 tmRNA 的报道，并且该纯化证明固相 DNA 探针法适用于含量低于 tRNA 的非编码 RNA。此外，如果长非编码 RNA 被特异性切割且可以用固相 DNA 探针法纯化片段，则可以分析长非编码 RNA 中的修饰核苷。因此，我们设计了一种脱氧核酶（DNAzyme）来特异性切割 16S rRNA，检查了最佳条件并纯化了得到的 RNA 片段。互补 DNA 的测序和质谱分析表明，纯化的 RNA 与 16S rRNA 的靶向片段相对应。因此，DNAzyme 切割与固相 DNA 探针方法相结合的纯化可以成为分析长非编码 RNA 中修饰核苷的有用技术。
