Chon Hyongi, Tsunaka Yasuo, Haruki Mitsuru, Morikawa Masaaki, Kanaya Shigenori
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Japan.
Protein Eng. 2002 Aug;15(8):683-8. doi: 10.1093/protein/15.8.683.
A series of DNA-linked RNases H, in which the 15-mer DNA is cross-linked to the Thermus thermophilus RNase HI (TRNH) variants at positions 135, 136, 137 and 138, were constructed and analyzed for their abilities to cleave the complementary 15-mer RNA. Of these, that with the DNA adduct at position 135 most efficiently cleaved the RNA substrate, indicating that position 135 is the most appropriate cross-linking site among those examined. To examine whether DNA-linked RNase H also site-specifically cleaves a highly structured natural RNA, DNA-linked TRNHs with a series of DNA adducts varying in size at position 135 were constructed and analyzed for their abilities to cleave MS2 RNA. These DNA adducts were designed such that DNA-linked enzymes cleave MS2 RNA at a loop around residue 2790. Of the four DNA-linked TRNHs with the 8-, 12-, 16- and 20-mer DNA adducts, only that with the 16-mer DNA adduct efficiently and site-specifically cleaved MS2 RNA. Primer extension revealed that this DNA-linked TRNH cleaved MS2 RNA within the target sequence.
构建了一系列DNA连接的核糖核酸酶H，其中15聚体DNA在135、136、137和138位与嗜热栖热菌核糖核酸酶HI（TRNH）变体交联，并分析了它们切割互补15聚体RNA的能力。其中，在135位带有DNA加合物的酶最有效地切割了RNA底物，表明135位是所检测位点中最合适的交联位点。为了研究DNA连接的核糖核酸酶H是否也能位点特异性切割高度结构化的天然RNA，构建了在135位带有一系列大小不同的DNA加合物的DNA连接的TRNH，并分析了它们切割MS2 RNA的能力。这些DNA加合物的设计使得DNA连接的酶在2790残基附近的环处切割MS2 RNA。在带有8聚体、12聚体、16聚体和20聚体DNA加合物的四种DNA连接的TRNH中，只有带有16聚体DNA加合物的酶能有效且位点特异性地切割MS2 RNA。引物延伸显示，这种DNA连接的TRNH在靶序列内切割MS2 RNA。
