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Protein Engineering, Vol. 15, No. 8, 683-688, August 2002
© 2002 Oxford University Press

Site-specific cleavage of MS2 RNA by a thermostable DNA-linked RNase H

Hyongi Chon, Yasuo Tsunaka, Mitsuru Haruki, Masaaki Morikawa and Shigenori Kanaya1

Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2–1 Yamadaoka, Suita, Osaka 565-0871, Japan

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.


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