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Site-specific cleavage of MS2 RNA by a thermostable DNA-linked RNase H
Hyongi Chon1, Yasuo Tsunaka, Mitsuru Haruki
1Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Japan.
Abstract:
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.
Insights
Researchers engineered DNA-linked Thermus thermophilus RNase HI (TRNH) enzymes. The DNA adduct at position 135 on TRNH demonstrated optimal RNA cleavage, enabling site-specific targeting of MS2 RNA.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- RNases H are enzymes that degrade RNA.
- Thermus thermophilus RNase HI (TRNH) is a specific type of RNase H.
- Modifying enzymes with DNA adducts can alter their activity and specificity.
Purpose of the Study:
- To investigate the optimal site for DNA cross-linking on TRNH to enhance RNA cleavage.
- To determine if DNA-linked TRNH can achieve site-specific cleavage of a complex natural RNA like MS2 RNA.
- To evaluate the effect of DNA adduct size on cleavage efficiency and specificity.
Main Methods:
- Construction and analysis of DNA-linked TRNH variants with cross-links at different positions (135-138).
- Assessment of RNA cleavage activity using a 15-mer RNA substrate.
- Design and synthesis of TRNH with DNA adducts of varying lengths (8, 12, 16, 20-mer) at position 135.
- Testing cleavage of MS2 RNA and site-specificity using primer extension assays.
Main Results:
- DNA cross-linking at position 135 of TRNH yielded the highest efficiency in cleaving a complementary 15-mer RNA.
- Among DNA-linked TRNHs with varying adduct sizes at position 135, only the 16-mer DNA adduct variant efficiently and site-specifically cleaved MS2 RNA.
- Primer extension confirmed that the 16-mer DNA-linked TRNH cleaved MS2 RNA at the intended loop region around residue 2790.
Conclusions:
- Position 135 is the most effective site for DNA cross-linking on TRNH for RNA cleavage.
- DNA-linked TRNH, particularly with a 16-mer adduct, can achieve precise, site-specific cleavage of structured natural RNAs.
- This engineered DNA-linked TRNH holds potential for targeted RNA manipulation in molecular biology applications.