Kumazawa Y, Yokogawa T, Tsurui H, Miura K, Watanabe K
Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan.
Nucleic Acids Res. 1992 May 11;20(9):2223-32. doi: 10.1093/nar/20.9.2223.
In the course of developing a method to purify a single tRNA species efficiently, we have examined hybridization efficiencies between some tRNAs and short oligodeoxyribonucleotide probes both by the filter and solution hybridization methods without denaturants. The hybridization efficiencies varied considerably among probes which are complementary to different regions of the tRNAs, although there was little efficiency variation in the probes toward DNA substrates including the same nucleotide sequence. This efficiency variation was shown to be due to tRNA-specific higher-order structures as well as a hypermodified nucleotide in the anticodon loop. Characterization of the tRNA-probe hybrids by both nondenaturing gel electrophoresis and chemical modification showed the existence of two stable hybridizing states as a function of ionic strength. Our results indicate that RNA molecules with a number of intramolecular base pairings are able to form stable hybrids with complementary sequences under nondenaturing conditions. On the basis of these data, an appropriate probe was designed to successfully purify yeast tRNA(Phe) by making a tRNA(Phe)-probe hybrid, which has a longer retention time in hydroxyapatite high performance liquid chromatography than the tRNA(Phe) itself.
在开发一种高效纯化单一tRNA种类的方法过程中，我们通过滤膜杂交法和无变性剂的溶液杂交法，研究了一些tRNA与短寡脱氧核糖核苷酸探针之间的杂交效率。尽管针对包含相同核苷酸序列的DNA底物的探针效率变化很小，但与tRNA不同区域互补的探针之间的杂交效率差异很大。这种效率差异被证明是由于tRNA特异性的高级结构以及反密码子环中的超修饰核苷酸。通过非变性凝胶电泳和化学修饰对tRNA-探针杂交体进行表征，结果表明存在两种随离子强度变化的稳定杂交状态。我们的结果表明，具有多个分子内碱基配对的RNA分子能够在非变性条件下与互补序列形成稳定的杂交体。基于这些数据，设计了一种合适的探针，通过制备tRNA（Phe）-探针杂交体成功纯化了酵母tRNA（Phe），该杂交体在羟基磷灰石高效液相色谱中的保留时间比tRNA（Phe）本身更长。
